Methods for treating osteoarthritis pain by administering a nerve growth factor antagonist and compositions containing the same

ABSTRACT

The invention concerns anti-NGF antibodies (such as anti-NGF antagonist antibodies), and polynucleotides encoding the same. The invention further concerns use of such antibodies and/or polynucleotides in the treatment and/or prevention of pain, including post-surgical pain, rheumatoid arthritis pain, and osteoarthritis pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.16/410,325, filed on May 13, 2019, which is a division of U.S. patentapplication Ser. No. 15/287,246, filed on Oct. 6, 2016, now U.S. Pat.No. 10,323,086, which is a division of U.S. patent application Ser. No.13/946,532, filed on Jul. 19, 2013, now U.S. Pat. No. 9,498,530, whichis a continuation-in-part of U.S. patent application Ser. No.13/302,264, filed on Nov. 22, 2011, now U.S. Pat. No. 9,212,222, whichis a continuation of U.S. patent application Ser. No. 12/618,896, filedon Nov. 16, 2009, now U.S. Pat. No. 8,088,384, which is a continuationof U.S. patent application Ser. No. 11/653,206, filed on Jan. 12, 2007,now U.S. Pat. No. 7,655,232, which is a continuation of U.S. patentapplication Ser. No. 10/745,775, filed on Dec. 24, 2003, now U.S. Pat.No. 7,449,616, which claims the priority benefit of U.S. ProvisionalApplication No. 60/436,905, filed on Dec. 24, 2002, and U.S. ProvisionalApplication No. 60/443,522, filed on Jan. 28, 2003, and U.S. ProvisionalApplication No. 60/510,006, filed on Oct. 8, 2003, all of which areincorporated herein in their entireties by reference. U.S. patentapplication Ser. No. 13/946,532 is also a division of U.S. patentapplication Ser. No. 11/911,305, filed on Apr. 11, 2006, now U.S. Pat.No. 8,540,990, which is a National Stage Application under 35 U.S.C. §371 of PCT/US2006/013921, filed on Apr. 11, 2006, which claims thepriority benefit of patent application U.S. Ser. No. 11/104,248, filedon Apr. 11, 2005, now U.S. Pat. No. 7,569,364, which is acontinuation-in-part of U.S. patent application Ser. No. 10/745,775,filed on Dec. 24, 2003, now U.S. Pat. No. 7,449,616, each of which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention concerns anti-NGF antibodies (such as anti-NGF antagonistantibodies). The invention further concerns use of antagonists such asantibodies in the treatment and/or prevention of pain, includingpost-surgical pain, rheumatoid arthritis pain, and osteoarthritis pain.

BACKGROUND OF THE INVENTION

Nerve growth factor (NGF) was the first neurotrophin to be identified,and its role in the development and survival of both peripheral andcentral neurons has been well characterized. NGF has been shown to be acritical survival and maintenance factor in the development ofperipheral sympathetic and embryonic sensory neurons and of basalforebrain cholinergic neurons. Smeyne et al., Nature 368:246-249 (1994)and Crowley et al., Cell 76:1001-1011 (1994). NGF up-regulatesexpression of neuropeptides in sensory neurons (Lindsay and Harmer,Nature 337:362-364 (1989)) and its activity is mediated through twodifferent membrane-bound receptors, the TrkA tyrosine kinase receptorand the p75 common neurotrophin receptor (sometimes termed “highaffinity” and “low affinity” NGF receptors, respectively). Chao et al.,Science 232:518-521 (1986). The p75 receptor is structurally related toother members of the tumor necrosis factor receptor family (Chao, etal., Science 232:518-521 (1986)). For review on NGF, see Huang et al.,Annu. Rev. Neurosci. 24:677-736 (2001); Bibel et al., Genes Dev.14:2919-2937 (2000). The crystal structure of NGF and NGF in complexwith the trkA receptor have been determined. See Nature 254:411 (1991);Nature 401:184-188 (1996).

In addition to its effects in the nervous system, NGF has beenincreasingly implicated in processes outside of the nervous system. Forexample, NGF has been shown to enhance vascular permeability (Otten, etal., Eur J Pharmacol. 106:199-201 (1984)), enhance T- and B-cell immuneresponses (Otten, et al., Proc. Natl. Acad. Sci. USA 86:10059-10063(1989)), induce lymphocyte differentiation and mast cell proliferationand cause the release of soluble biological signals from mast cells(Matsuda, et al., Proc. Natl. Acad. Sci. USA 85:6508-6512 (1988);Pearce, et al., J. Physiol. 372:379-393 (1986); Bischoff, et al., Blood79:2662-2669 (1992); Horigome, et al., J. Biol. Chem. 268:14881-14887(1993)). Although exogenously added NGF has been shown to be capable ofhaving all of these effects, it is important to note that it has onlyrarely been shown that endogenous NGF is important in any of theseprocesses in vivo (Torcia, et al., Cell. 85(3):345-56 (1996)).Therefore, it is not clear what that effect might be, if any, ofinhibiting the bioactivity of endogenous NGF.

NGF is produced by a number of cell types including mast cells (Leon, etal., Proc. Natl. Acad. Sci. USA 91:3739-3743 (1994)), B-lymphocytes(Torcia, et al., Cell 85:345-356 (1996), keratinocytes (Di Marco, etal., J. Biol. Chem. 268:22838-22846)), smooth muscle cells (Ueyama, etal., J. Hypertens. 11:1061-1065 (1993)), fibroblasts (Lindholm, et al.,Eur. J. Neurosci. 2:795-801 (1990)), bronchial epithelial cells (Kassel,et al., Clin, Exp. Allergy 31:1432-40 (2001)), renal mesangial cells(Steiner, et al., Am. J. Physiol. 261:F792-798 (1991)) and skeletalmuscle myotubes (Schwartz, et al., J Photochem. Photobiol. B66:195-200(2002)). NGF receptors have been found on a variety of cell typesoutside of the nervous system. For example, TrkA has been found on humanmonocytes, T- and B-lymphocytes and mast cells.

An association between increased NGF levels and a variety ofinflammatory conditions has been observed in human patients as well asin several animal models. These include systemic lupus erythematosus(Bracci-Laudiero, et al., Neuroreport 4:563-565 (1993)), multiplesclerosis (Bracci-Laudiero, et al., Neurosci. Lett. 147:9-12 (1992)),psoriasis (Raychaudhuri, et al., Acta Derm. l'enereol. 78:84-86 (1998)),arthritis (Falcim, et al., Ann. Rheum. Dis. 55:745-748 (1996)),interstitial cystitis (Okragly, et al., J. Urology 161:438-441 (1999))and asthma (Braun, et al., Eur. J Immunol. 28:3240-3251 (1998)).

Consistently, an elevated level of NGF in peripheral tissues isassociated with hyperalgesia and inflammation and has been observed in anumber of forms of arthritis. The synovium of patients affected byrheumatoid arthritis expresses high levels of NGF while in non-inflamedsynovium NGF has been reported to be undetectable (Aloe, et al., Arch.Rheum. 35:351-355 (1992)). Similar results were seen in rats withexperimentally induced rheumatoid arthritis (Aloe, et al., Clin. Exp.Rheumatol. 10:203-204 (1992)). Elevated levels of NGF have been reportedin transgenic arthritic mice along with an increase in the number ofmast cells (Aloe, el al., Int. J. Tissue Reactions-Exp. Clin. Aspects15:139-143 (1993)). PCT Publication No. WO 02/096458 discloses use ofanti-NGF antibodies of certain properties in treating various NGFrelated disorders such as inflammatory condition (e.g., rheumatoidarthritis). It has been reported that a purified anti-NGF antibodyinjected into arthritic transgenic mice carrying the human tumornecrosis factor-α (TNF-α) gene caused reduction in the number of mastcells, as well as a decrease in histamine and substance P levels withinthe synovium of arthritis mice (Aloe et al., Rheumatol. Int. 14: 249-252(1995)). It has been shown that exogenous administration of a NGFantibody reduced the enhanced level of TNF-α occurring in arthritic mice(Manni et al., Rheumatol. Int. 18: 97-102 (1998)).

Also, increased expression of NGF and high affinity NGF receptor (TrkA)was observed in human osteoarthritis chondrocytes (Iannone et al.,Rheumatology 41:1413-1418 (2002)).

Rodent anti-NGF antagonist antibodies have been reported. See, e.g.,Hongo et al, Hybridoma (2000) 19(3):215-227; Ruberti et Al. (1993) Cell.Molec. Neurobiol. 13(5): 559-568. However, when rodent antibodies areused therapeutically in humans, a human anti-murine antibody responsedevelops in significant numbers of treated individuals. In addition,effector functions of mouse antibodies have proven to be less efficientin the human context. Thus, there is a serious need for anti-NGFantagonist antibodies, including humanized anti-NGF antagonistantibodies.

All references, publications, and patent applications disclosed hereinare hereby incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The invention disclosed herein concerns antibodies to nerve growthfactor.

In another aspect, the invention is a humanized and affinity maturedantibody, E3, which specifically binds human and rodent nerve growthfactor (“NGF”). The amino acid sequences of the heavy chain and lightchain variable regions of E3 are shown in FIGS. 1A (SEQ ID NO:1) and 1B(SEQ ID NO:2), respectively. The CDR portions of antibody E3 (includingChothia and Kabat CDRs) are diagrammatically depicted in FIGS. 1A and1B. The amino acid sequences of E3 heavy and light chains, and of theindividual extended CDRs are also shown below (See, “antibodysequences”, below).

In another aspect, the invention is an antibody comprising a fragment ora region of the antibody E3 (interchangeably termed “E3” herein). In oneembodiment, the fragment is a light chain of the antibody E3 as shown inFIG. 1B. In another embodiment, the fragment is a heavy chain of theantibody E3 as shown in FIG. 1A. In yet another embodiment, the fragmentcontains one or more variable regions from a light chain and/or a heavychain of the antibody E3. In yet another embodiment, the fragmentcontains one or more complementarity determining regions (CDRs) from alight chain and/or a heavy chain of the antibody E3 as shown in FIGS. 1Aand 1B.

In another aspect, the invention is an antibody comprising a light chainthat is encoded by a polynucleotide that is produced by a host cell witha deposit number of ATCC No. PTA-4893 or ATCC No. PTA-4894. In anotheraspect, the invention is an antibody comprising a heavy chain that isencoded by a polynucleotide that is produced by a host cell with adeposit number of ATCC No. PTA-4895. In another aspect, the invention isan antibody comprising (a) a light chain that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4894 or ATCC No. PTA-4893; and (b) a heavy chain that isencoded by a polynucleotide that is produced by a host cell with adeposit number of ATCC No. PTA-4895 (for convenience herein, thepolynucleotide(s) produced by a deposited host cell are referred to ashaving a deposit number of ATCC NOs PTA-4894, PTA-4893 and PTA-4895). Inanother aspect, the invention is an antibody comprising a light chainvariable region of a light chain that is encoded by a polynucleotidethat is produced by a host cell with a deposit number of ATCC No.PTA-4894 or ATCC No. PTA-4893. In another aspect, the invention is anantibody comprising a heavy chain variable region of a heavy chain thatthat is encoded by a polynucleotide that is produced by a host cell witha deposit number of ATCC No. PTA-4895. In another aspect, the inventionis an antibody comprising (a) a light chain variable region of a lightchain that is encoded by a polynucleotide that is produced by a hostcell with a deposit number of ATCC No. PTA-4894 or ATCC No. PTA-4893,and (b) a heavy chain variable region of a heavy chain that that isencoded by a polynucleotide that is produced by a host cell with adeposit number of ATCC No. PTA-4895. In still another aspect, theinvention is an antibody comprising one or more CDR(s) encoded by (a) apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4894; and/or (b) a heavy chain that is encoded by apolynucleotide that is produced by a host cell with a deposit number ofATCC No. PTA-4895.

In some embodiments, the antibody comprises the human heavy chain IgG2aconstant region. In some embodiments the antibody comprises the humanlight chain kappa constant region. In some embodiments, the antibodycomprises a modified constant region, such as a constant region that isimmunologically inert, e.g., does not trigger complement mediated lysis,or does not stimulate antibody-dependent cell mediated cytotoxicity(ADCC). In other embodiments, the constant region is modified asdescribed in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No.PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In stillother embodiments, the antibody comprises a human heavy chain IgG2aconstant region comprising the following mutations: A330P331 to S330S331(amino acid numbering with reference to the wildtype IgG2a sequence).Eur. J. Immunol. (1999) 29:2613-2624.

In another aspect, the invention provides polypeptides (which may or maynot be an antibody) comprising any one or more of the following: a) oneor more CDR(s) of antibody E3 shown in FIGS. 1A and 1B; b) CDR H3 fromthe heavy chain of antibody E3 shown in FIG. 1A; c) CDR L3 from thelight chain of antibody E3 shown in FIG. 1B; d) three CDRs from thelight chain of antibody E3 shown in FIG. 1B; e) three CDRs from theheavy chain of antibody E3 shown in FIG. 1A; and f) three CDRs from thelight chain and three CDRs from the heavy chain, of antibody E3 shown inFIGS. 1A and 1B. The invention further provides polypeptides (which mayor may not be an antibody) comprising any one or more of the following:a) one or more (one, two, three, four, five, or six) CDR(s) derived fromantibody E3 shown in FIGS. 1A and 1B; b) a CDR derived from CDR H3 fromthe heavy chain of antibody E3 shown in FIG. 1A; and/or c) a CDR derivedfrom CDR L3 from the light chain of antibody E3 shown in FIG. 1B. Insome embodiments, the CDRs may be Kabat CDRs, Chothia CDRs, or acombination of Kabat and Chothia CDRs (termed “extended” or “combined”CDRs herein). In some embodiments, polypeptides (such as an antibody)bind NGF (such as human NGF). In some embodiments, the polypeptidescomprise any of the CDF configurations (including combinations,variants, etc.) described herein.

In one aspect, the invention provides polypeptides (such as anantibody), which comprise a heavy chain variable region comprising SEQID NO:9, wherein I34 is S, L, V A, or I; and N35 is substituted with N,T or S. For convenience herein, “substituted” or “is” in this context orreference to an amino acid refers to choices of amino acid(s) for agiven position. As is clear, the substitution, or choice, may be theamino acid depicted in a SEQ ID or Figure.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a heavy chain variable region comprising SEQ IDNO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is Lor V.

In another aspect, the invention provides polypeptides (such as anantibody) which comprises a heavy chain variable region comprising SEQID NO: 11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is D, N, or G; and wherein Y110 is Y, K, S, R or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a heavy chain variable region comprising SEQ IDNO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is anyamino acid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a heavy chain variable region comprising SEQ IDNO: 11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S,A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G; and whereinY110 is any amino acid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, orQ.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO: 13, wherein 151 is I, T, V or A; and S56 is S or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO:14, wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Yor R.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising SEQ IDNO:14, wherein S91 is S or E; K92 is any amino acid; T93 is any aminoacid; and wherein Y96 is Y or R.

In one aspect, the invention provides polypeptides (such as anantibody), which comprise an amino acid sequence shown in SEQ ID NO:9,wherein I34 is S, L, V A, or I; and N35 is N, Tor S.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO:10,wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is D, N, or G; and wherein Y110 is Y, K, S, R or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO:11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is any aminoacid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 11,wherein G98 is G, S, A, C, V, N, D, or T; wherein G99 is G, S, A, C, V,N, D, or T; wherein Y100 is Y, L, or R; wherein Y101 is Y or W; whereinG103 is G, A, or S; wherein T104 is T or S; wherein S105 is S, A, or T;wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is For W; wherein D109 is S, A, C, G, D, N, T, or G; and wherein Y110 is anyamino acid.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO:12,wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO: 13,wherein 151 is I, T, V or A; and S56 is S or T.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO:14,wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO:14,wherein S91 is S or E; K92 is any amino acid; T93 is any amino acid; andwherein Y96 is Y or R.

In another aspect, the invention provides polypeptides (such anantibodies, including humanized antibodies) which comprise a heavy chainvariable region comprising the CDR1 region of SEQ ID NO:9, wherein I34is S, L, V A, or I; and N35 is N, T or S; the CDR2 region of SEQ IDNO:10, wherein M50 is M, I, G, Q, S, or L; A62 is A, or S; and L63 is Lor V; and the CDR3 region of SEQ ID NO:11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G;wherein Y110 is Y, K, S, R or T. In some embodiments, the heavy chainvariable region comprises the CDR3 region of SEQ ID NO:11, wherein Y100is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G, A, or S;wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y,R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109is S, A, C, G, D, N, T, or G; wherein Y110 is any amino acid. In otherembodiments, the heavy chain variable region comprises the CDR3 regionof SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, or T; wherein G99is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R; wherein Y101is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; whereinS105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y orF; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T, or G;and wherein Y110 is any amino acid. In some embodiments, the polypeptide(such as an antibody) further comprises an antibody light chain variableregion.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise a light chain variable region comprising theCDR1 region of SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, orY; and H32 is H, N, or Q; the CDR2 region of SEQ ID NO:13, wherein 151is I, T, V or A; and S56 is S or T; and the CDR3 region of SEQ ID NO:14,wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.In some embodiments, the light chain variable region comprises the CDR3region of SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid;T93 is any amino acid; and wherein Y96 is Y or R. In some embodiments,the polypeptide (such as an antibody) further comprises an antibodyheavy chain.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise (a) a heavy chain variable region comprisingthe CDR1 region of SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35is N, T or S; the CDR2 region of SEQ ID NO:10, wherein M50 is M, I, G,Q, S, or L; A62 is A, or S; and L63 is L or V; and the CDR3 region ofSEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is D, N, or G; wherein Y110 is Y, K, S, Ror T; and (b) a light chain variable region comprising the CDR1 regionof SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, or Y; and H32 isH, N, or Q; the CDR2 region of SEQ ID NO:13, wherein 151 is I, T, V orA; and S56 is S or T; and the CDR3 region of SEQ ID NO:14, wherein S91is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R. In someembodiments, the light chain variable region comprises the CDR3 regionof SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 isany amino acid; and wherein Y96 is Y or R. In some embodiments, theheavy chain variable region comprises the CDR3 region of SEQ ID NO:11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; wherein Y110 is any aminoacid. In other embodiments, the heavy chain variable region comprisesthe CDR3 region of SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, orT; wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid. In some embodiments, thepolypeptide further comprises an antibody light chain.

In another aspect, the invention provides polypeptides (such anantibody, including a humanized antibody) which comprise an amino acidsequence shown in SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35is N, T or S; an amino acid sequence shown in SEQ ID NO:10, wherein M50is M, I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and an aminoacid sequence shown in SEQ ID NO: 11, wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G;wherein Y110 is Y, K, S, R or T. In some embodiments, the polypeptidecomprises an amino acid sequence shown in SEQ ID NO: 11, wherein Y100 isY, L, or R; and wherein Y101 is Y or W; wherein G103 is G, A, or S;wherein T104 is T or S; wherein S105 is S, A, or T; wherein Y106 is Y,R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W; wherein D109is S, A, C, G, D, N, T, or G; and wherein Y110 is any amino acid. Inother embodiments, the polypeptide comprises an amino acid sequenceshown in SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, or T; whereinG99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R; whereinY101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S;wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D, N, T,or G; and wherein Y110 is any amino acid. In some embodiments, thepolypeptide (such as an antibody) further comprises an antibody lightchain variable region.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise an amino acid sequence shown in SEQ ID NO:12,wherein S26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; anamino acid sequence shown in SEQ ID NO:13, wherein 151 is I, T, V or A;and S56 is S or T; and an amino acid sequence shown in SEQ ID NO:14,wherein S91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R.In some embodiments, the polypeptide comprises an amino acid sequenceshown in SEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93is any amino acid; and wherein Y96 is Y or R. In some embodiments, thepolypeptide (such as an antibody) further comprises an antibody heavychain variable region.

In another aspect, the invention provides polypeptides (such as anantibody) which comprise (a) an amino acid sequence shown in SEQ IDNO:9, wherein I34 is S, L, V A, or I; and N35 is N, T or S; an aminoacid sequence shown in SEQ ID NO:10, wherein M50 is M, I, G, Q, S, or L;A62 is A, or S; and L63 is L or V; and an amino acid sequence shown inSEQ ID NO:11, wherein Y100 is Y, L, or R; wherein Y101 is Y or W;wherein G103 is G, A, or S; wherein T104 is T or S; wherein S105 is S,A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y or F; whereinF108 is F or W; wherein D109 is D, N, or G; and wherein Y110 is Y, K, S,R or T; and (b) an amino acid sequence shown in SEQ ID NO:12, whereinS26 is S or F; D28 is D, S, A, or Y; and H32 is H, N, or Q; an aminoacid sequence shown in SEQ ID NO:13, wherein 151 is I, T, V or A; andS56 is S or T; and an amino acid sequence shown in SEQ ID NO:14, whereinS91 is S or E; K92 is K, H, R, or S; and wherein Y96 is Y or R. In someembodiments, the polypeptide comprises an amino acid sequence shown inSEQ ID NO:14, wherein S91 is S or E; K92 is any amino acid; T93 is anyamino acid; and wherein Y96 is Y or R. In some embodiments, thepolypeptide comprises an amino acid sequence shown in SEQ ID NO:11,wherein Y100 is Y, L, or R; wherein Y101 is Y or W; wherein G103 is G,A, or S; wherein T104 is T or S; wherein S105 is S, A, or T; whereinY106 is Y, R, T, or M; wherein Y107 is Y or F; wherein F108 is F or W;wherein D109 is S, A, C, G, D, N, T, or G; wherein Y110 is any aminoacid. In other embodiments, the polypeptide comprises an amino acidsequence shown in SEQ ID NO:11, wherein G98 is G, S, A, C, V, N, D, orT; wherein G99 is G, S, A, C, V, N, D, or T; wherein Y100 is Y, L, or R;wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is T orS; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is S, A, C, G, D,N, T, or G; and wherein Y110 is any amino acid. In some embodiments, thepolypeptide further comprises an antibody light chain variable region.

In another aspect, the invention provides polypeptide (such asantibodies) comprising a heavy chain variable region comprising: (a) aCDR1 region of SEQ ID NO:9, wherein I34 is S, L, V A, or I; and N35 issubstituted with N, T or S; (b) a CDR2 region of SEQ ID NO:10, whereinM50 is I, G, Q, S, or L; A62 is A, or S; and L63 is L or V; and (c) aCDR3 region of SEQ ID NO: 11, wherein Y100 is Y, L, or R; wherein Y101is Y or W; wherein G103 is G, A, or S; wherein T104 is T or S; whereinS105 is S, A, or T; wherein Y106 is Y, R, T, or M; wherein Y107 is Y orF; wherein F108 is F or W; wherein D109 is D, N, or G; and wherein Y110is Y, K, S, R or T; wherein the antibody binds NGF.

In another aspect, the invention provides polypeptides (such asantibodies) comprising a light chain variable region comprising: (a) aCDR1 region of SEQ ID NO:12, wherein S26 is S or F; D28 is D, S, A, orY; and H32 is H, N, or Q; (b) a CDR2 region of SEQ ID NO: 13, wherein151 is I, T, V or A; and S56 is S or T; and (c) a CDR3 region of SEQ IDNO:14, wherein K92 is K, H, R, or S; and wherein Y96 is Y or R; whereinthe antibody binds NGF.

In another aspect, the invention provides polypeptides (such asantibodies) comprising (a) a heavy chain variable region comprising: (i)a CDR1 region of SEQ ID NO:9, wherein I34 is substituted with S, L, V A,or I; and N35 is substituted with N, T or S; (ii) a CDR2 region of SEQID NO:10, wherein M50 is I, G, Q, S, or L; A62 is A, or S; and L63 is Lor V; and (iii) a CDR3 region of SEQ ID NO: 11, wherein Y100 is Y, L, orR; wherein Y101 is Y or W; wherein G103 is G, A, or S; wherein T104 is Tor S; wherein S105 is S, A, or T; wherein Y106 is Y, R, T, or M; whereinY107 is Y or F; wherein F108 is F or W; wherein D109 is D, N, or G;wherein Y110 is Y, K, S, R or T; and (b) a light chain variable regioncomprising: (i) a CDR1 region of SEQ ID NO:12, wherein S26 is S or F;D28 is D, S, A, or Y; and H32 is H, N, or Q; (ii) a CDR2 region of SEQID NO: 13, wherein 151 is I, T, V or A; and S56 is S or T; and (iii) aCDR3 region of SEQ ID NO:14, wherein S91 is S or E; K92 is K, H, R, orS; and wherein Y96 is Y or R; wherein the antibody binds NGF.

Unless otherwise noted, choice (e.g., substitution) of an amino acid inone location is independently selected from selection of an amino acidin any other location.

In some embodiments, polynucleotides (such as an antibody) bind NGF(such as human NGF). In some embodiments, the polypeptides comprise anyof the CDR configurations (including combinations, variations, etc.)described herein.

As is evident from the description herein, the variable region numberingused herein is sequential numbering. One of skill in the art readilyunderstands that a number of antibody numbering systems exist (such asKabat and Chothia numbering), and how to convert sequential numberinginto another numbering system, such as Kabat numbering or Chothianumbering.

In another aspect, the invention provides a polypeptide (such as anantibody) comprising an amino acid sequence (such as a CDR3 sequence)selected from SEQ ID NO:46 or 50. In still other embodiments, thepolypeptide further comprises one or more of the amino acid sequencesshown in SEQ ID NOS:3, 4, 5, 6, 7, and 8. In still other embodiments,the polypeptide further comprises one of more of the amino acidsequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.

In another aspect, the invention provides a polypeptide (such as anantibody) comprising an amino acid sequence (such as a CDR region, suchas a CDRH1 and/or CDR H2 region) selected from (a) SEQ ID NOS:28 and/or29; (b) SEQ ID NOS:30 and/or 31; (c) SEQ ID NOS:32 and/or 33; (d) SEQ IDNOS:34 and/or 35; (e) SEQ ID NOS:36 and/or 37; (f) SEQ ID NOS:38 and/or39; and (g) SEQ ID NOS:40 and 41. In some embodiments, the polypeptidecomprises an amino acid sequence (such as a CDR H1 region) selected fromSEQ ID NOS:28, 30, 32, 34, 36, 38, and 40. In some embodiments, thepolypeptide comprises an amino acid sequence (such as a CDR H2 region)selected from SEQ ID NOS:29, 31, 33, 35, 37, 39 and 41. In still otherembodiments, the polypeptide further comprises one or more of the aminoacid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, and 8. In still otherembodiments, the polypeptide further comprises one of more of the aminoacid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.

In another aspect, the invention provides a polypeptide (such as anantibody) comprising an amino acid sequence (such as a CDR region, suchas a CDRL1 and/or CDR L2 region) selected from (a) SEQ ID NOS:18 and/or19; (b) SEQ ID NOS:20 and/or 21; and (c) SEQ ID NOS:22 and/or 23. Insome embodiments, the polypeptide comprises an amino acid sequence (suchas a CDR L1 region) selected from SEQ ID NOS:18, 20, and 22. In someembodiments, the polypeptide comprises an amino acid sequence (such as aCDR L2 region) selected from SEQ ID NOS:19, 21, and 23. In still otherembodiments, the polypeptide further comprises one or more of the aminoacid sequences shown in SEQ ID NOS:3, 4, 5, 6, 7, 8. In still otherembodiments, the polypeptide further comprises one of more of the aminoacid sequences shown in SEQ ID NOS:9, 10, 11, 12, 13, 14, and 15.

In another aspect, the invention provides a polypeptide (such as anantibody) comprising an amino acid sequence (such as a CDR region, suchas a CDRL3 and/or CDR H3 region) selected from (a) SEQ ID NOS:51 and/or52; (b) SEQ ID NOS:55 and/or 56; (c) SEQ ID NOS:57 and/or 58; (c) SEQ IDNOS:59 and/or 60; (d) SEQ ID NOS:61 and/or 62; (e) SEQ ID NOS:63 and/or64. In some embodiments, the polypeptide comprises an amino acidsequence (such as a CDR L3 region) selected from SEQ ID NOS:51, 55, 57,59, 61, and 63. In some embodiments, the polypeptide comprises an aminoacid sequence (such as a CDR H3 region) selected from SEQ ID NOS:52, 56,58, 60, 62, and 64. In still other embodiments, the polypeptide furthercomprises an amino acid sequence shown in one or more of SEQ ID NOS:18,19, 30 and 31. In still other embodiments, the polypeptide furthercomprises one or more of the amino acid sequences shown in SEQ ID NOS:3,4, 5, 6, 7, and 8. In still other embodiments, the polypeptide furthercomprises one of more of the amino acid sequences shown in SEQ ID NOS:9,10, 11, 12, 13, 14, and 15.

In another aspect, the invention provides a polypeptide (such as anantibody) comprising one or more of an amino acid sequence (such as aCDR region) shown in SEQ ID NOS:61, 63, 18, 19, 30 and 31.

In one aspect, the invention provides an anti-NGF antibody (such as anantagonist antibody) that binds NGF (such as human NGF) with a highaffinity. In some embodiments, high affinity is (a) binding NGF with aK_(D) of less than about 2 nM (such as any of about 1 nM, 800 pM, 600pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, or less),and/or a k_(off) of slower than about 6×10⁵ s⁻¹); and/or (b) inhibiting(reducing, and/or blocking) human NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 (in the presence of about 15 pM of NGF)of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10pM, or less; and/or (c) inhibiting (reducing, and/or blocking) humanNGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50(in the presence of about 1.5 pM of NGF) of about any of 50 pM, 40 pM,30 pM, 10 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, or less; and/or (d)inhibiting (reducing, and/or blocking) rat NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 (in the presence of about 15pM of NGF) of about any of 150 pM, 125 pM, 100 pM, 80 pM, 60 pM, 40 pM,30 pM, 20 pM, 10 pM, 5 pM, or less; and/or (e) inhibiting (reducing,and/or blocking) rat NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 (in the presence of about 1.5 pM of NGF) of aboutany of 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM,or less; and/or (f) and/or bind NGF with higher affinity than does thetrkA receptor.

In another aspect, the invention provides polypeptides (such as anantibody), wherein the polypeptides (a) bind NGF (such as human NGF)with a K_(D) of less than about 2 nM (such as any of about 1 nM, 800 pM,600 pM, 400 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, orless), and/or a k_(off) of slower than about 6×10⁵ s⁻¹); and/or (b)inhibit human NGF-dependent survival of mouse E13.5 trigeminal neuronswith an IC50 (in the presence of about 15 pM of NGF) of about any of 200pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM, 10 pM, or less; and/or(c) inhibit human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 (in the presence of about 1.5 pM of NGF) of aboutany of 50 pM, 40 pM, 30 pM, 10 pM, 20 pM, 10 pM, 5 pM, 2 pM, 1 pM, orless; and/or bind NGF with higher affinity than does the trkA receptor.In some embodiments, the polypeptides (a) bind NGF with a K_(D) of lessthan about 2 nM; and/or (b) inhibit human NGF-dependent survival ofmouse E13.5 trigeminal neurons with an IC50 of about 100 pM or less,wherein the IC50 is measured in the presence of about 15 pM NGF; and/or(c) inhibit human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 10 pM or less, wherein the IC50 ismeasured in the presence of about 1.5 pM of NGF, wherein the IC50 ismeasured in the presence of about 15 pM NGF. In some embodiments, thepolypeptides (a) bind NGF with a K_(D) of less than about 100 pM; and/or(b) inhibit human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 20 pM or less, wherein the IC50 ismeasured in the presence of about 15 pM NGF; and/or (c) inhibit humanNGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 ofabout 2 pM or less, wherein the IC50 is measured in the presence ofabout 1.5 pM of NGF.

As is evident from the description herein, specifically excluded fromthe invention are polypeptide embodiments consisting of the identicalamino acid sequence to an amino acid sequence of mouse monoclonalantibody, 911. The extended CDR sequences of Mab 911 are shown in FIGS.1A and 1, and in SEQ ID NOS:9-14.

In some embodiments, the invention provides any of the abovepolypeptides or antibodies, further wherein the polypeptide (such as anantibody) is isolated. In some embodiments, the polypeptide (such as anantibody) is substantially purified. In still other embodiments, thepolypeptide (such as an antibody) is affinity matured. In otherembodiments, the antibody is an antagonist antibody. In someembodiments, the polypeptide (such as an antibody) comprises humanframework sequences. In still other embodiments, the polypeptide (suchas an antibody) comprises one or more non-human framework residues. Insome embodiments, the polypeptide (such as an antibody) binds NGF (suchas human NGF) with a K_(D) of 2 nM or less. In some embodiments, thepolypeptide comprises one or more (such as 2, 3, 4, 5, 6, 7, 8, or more)human amino acid substitutions relative to a non-human amino acidsequence (such as a variable region sequence, such as a CDR sequence,such as a framework sequence). In some embodiments, the polypeptidecomprises at least 1, at least 2, or more such as at least 3, 4, 5, 6,or more amino acid substitutions relative to a parent polypeptide aminoacid sequence (such as an antibody 911 amino acid sequence, such as anyone or more of SEQ ID NOs 9-14). In some embodiments, the bindingaffinity of the antibody has been altered (in some embodiments,increased) relative to a parent antibody (such as Mab 911) affinity. Instill other embodiments, the binding affinity of the antibody is lowerthan the binding affinity of trkA receptor for NGF (such as human NGF).In some embodiments, the polypeptides may be antibodies. In someembodiments, the antibodies are human antibodies. In other embodiments,the antibodies are humanized antibodies. In still other embodiments, theantibodies are monoclonal antibodies. In some embodiments, the antibodyis an affinity matured antibody.

The invention provides polynucleotides (including isolatedpolynucleotide) comprising polynucleotides encoding any of theembodiments above.

In another aspect, the invention provides an isolated polynucleotidecomprising a polynucleotide encoding a fragment or a region of theantibody E3 (interchangeably termed “E3” herein). In one embodiment, thefragment is a light chain of the antibody E3 as shown in FIG. 1B. Inanother embodiment, the fragment is a heavy chain of the antibody E3 asshown in FIG. 1A. In yet another embodiment, the fragment contains oneor more variable regions from a light chain and/or a heavy chain of theantibody E3. In yet another embodiment, the fragment contains one ormore complementarity determining regions (CDRs) from a light chainand/or a heavy chain of the antibody E3 as shown in FIGS. 1A and 1B.

In another aspect, the invention is an isolated polynucleotidecomprising a polynucleotide that encodes for antibody E3. In someembodiments, the polynucleotide comprises either or both of thepolynucleotide shown in FIGS. 2 and 3.

In another aspect, the invention is an isolated polynucleotide thatencodes for an E3 light chain with a deposit number of ATCC No. PTA-4893or ATCC No. PTA-4894. In another aspect, the invention is an isolatedpolynucleotide that encodes for an E3 heavy chain with a deposit numberof ATCC No. PTA-4895. In yet another aspect, the invention is anisolated polynucleotide comprising (a) a variable region encoded in thepolynucleotide with a deposit number of ATCC No. PTA-4893 or PTA-4894and (b) a variable region encoded in the polynucleotide with a depositnumber of ATCC No. PTA-4895. In another aspect, the invention is anisolated polynucleotide comprising (a) one or more CDR encoded in thepolynucleotide with a deposit number of ATCC No. PTA-4893 or PTA-4894;and/or (b) one or more CDR encoded in the polynucleotide with a depositnumber of ATCC No. PTA-4895.

In another aspect, the invention provides polynucleotides encoding anyof the antibodies (including antibody fragments) or polypeptidesdescribed herein.

In another aspect, the invention provides vectors (including expressionand cloning vectors) and host cells comprising any of the polynucleotidedisclosed herein.

As is evident from the description herein, specifically included fromthe invention are polynucleotide embodiments consisting of the identicalpolynucleotide sequence to a polynucleotide sequence of mouse monoclonalantibody, 911. The extended CDR sequences of Mab 911 are shown in FIGS.1A and 1B, and in SEQ ID NOS:9-14.

In another aspect, the invention is a host cell comprising apolynucleotide encoding E3 light chain and a polynucleotide encoding E3heavy chain, wherein the polynucleotide(s) encoding E3 light chain has adeposit number of ATCC No. PTA-4893 and/or ATCC No. PTA-4894, and thepolynucleotide encoding E3 heavy chain has a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell comprises polynucleotidecomprising (a) a variable region encoded in the polynucleotide with adeposit number of ATCC No. PTA-4893 or PTA-4894 and/or (b) a variableregion encoded in the polynucleotide with a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell comprises a polynucleotideencoding (a) one or more CDR encoded in the polynucleotide with adeposit number of ATCC No. PTA-4893 or PTA-4894; and/or (b) one or moreCDR encoded in the polynucleotide with a deposit number of ATCC No.PTA-4895. In some embodiments, the host cell is a mammalian cell.

In another aspect, the invention is a complex of NGF bound by antibodyE3. In another aspect, the complex is isolated. In another aspect, thecomplex is substantially purified.

In another aspect, the invention is a complex of NGF bound by any of theantibodies or polypeptides described herein. In another aspect, thecomplex is isolated. In another aspect, the complex is substantiallypurified.

In another aspect, the invention is a pharmaceutical compositioncomprising any of the polypeptides (including antibodies such asantibody E3) or polynucleotides described herein, such as pharmaceuticalcompositions comprising the antibody E3 or an antibody comprising afragment of the antibody E3, and a pharmaceutically acceptableexcipient.

In another aspect, the invention is a method of generating antibody E3comprising preparing a host cell comprising an expression vector thatencodes for antibody E3; culturing the host cell or progeny thereofunder conditions that allow production of antibody E3; and purifying theantibody E3. In some embodiments, the expression vector comprises one orboth of the polynucleotide sequences shown in FIGS. 2 and 3.

In another aspect, the invention is a method of generating antibody E3comprising expressing a polynucleotide encoding E3 light chain and apolynucleotide encoding E3 heavy chain in a suitable cell, wherein thepolynucleotide encoding E3 light chain has a deposit number of ATCC No.PTA-4893 and/or ATCC No. PTA-4894, and the polynucleotide encoding E3heavy chain has a deposit number of ATCC No. PTA-4895; generallyfollowed by recovering and/or isolating the antibody.

In another aspect, the invention provides methods of generating any ofthe polypeptides (such as antibodies) described herein by expressing oneor more polynucleotides encoding the antibody (which may be separatelyexpressed as a single light or heavy chain, or both a light and a heavychain may be expressed from one vector) in a suitable cell, generallyfollowed by recovering and/or isolating the antibody or polypeptides ofinterest.

In another aspect, the invention is a method of antagonizing NGF (suchas human NGF) biological activity using any of the polypeptides(including antibodies such as antibody E3) disclosed herein. In oneembodiment, the method comprises contacting human nerve growth factorwith any of the polypeptides (including antibody E3) described herein,whereby NGF activity (such as human nerve growth factor activity) isantagonized, reduced, blocked, or suppressed.

In another aspect, the invention is a method of detecting NGF using anyof the polypeptides (including antibodies, such as the antibody E3)described herein. The presence of NGF is detected by detecting a complexbetween NGF and any of the polypeptides described herein (such asantibody E3). The term “detection” as used herein includes qualitativeand/or quantitative detection (measuring levels) with or withoutreference to a control.

In another aspect, the invention is a method of treating pain byadministering an effective amount of a composition comprising theantibody E3 or any of the polypeptide (including antibody) orpolynucleotide embodiments described herein. In some embodiments, thepain is post-surgical pain.

In another aspect, the invention is a method for preventing or treatingrheumatoid arthritis pain in an individual by administering an effectiveamount of anti-NGF antagonist antibody to the individual. It has beenshown in accordance with the invention that an anti-NGF antagonistantibody is capable of inhibiting or blocking the pain associated withrheumatoid arthritis. In some embodiments, the pain is alleviated withinabout 24 hours after administering the anti-NGF antagonist antibody. Insome embodiments, the pain is alleviated within about 4 days afteradministering the anti-NGF antagonist antibody. In some embodiments, thepain is alleviated before observing or in the absence of an indicationof improvement of the inflammatory condition in the individual.

In another aspect, the invention provides methods for reducing incidenceof rheumatoid arthritis pain, ameliorating rheumatoid arthritis pain,suppressing rheumatoid arthritis pain, palliating rheumatoid arthritispain, and/or delaying the onset, development, or progression ofrheumatoid arthritis pain in an individual, said method comprisingadministering an effective amount of anti-NGF antagonist antibody to theindividual.

In another aspect, the invention provides methods for treatinginflammatory cachexia (weight loss) associated with rheumatoid arthritisin an individual comprising administering an effective amount of ananti-NGF antagonist antibody.

In another aspect, the invention is a method for preventing or treatingosteoarthritis pain in an individual by administering an effectiveamount of an antagonist of nerve growth factor (such as an anti-NGFantagonist antibody) to the individual.

In another aspect, the invention provides methods for reducing incidenceof osteoarthritis pain, ameliorating osteoarthritis pain, suppressingosteoarthritis pain, palliating osteoarthritis pain, and/or delaying theonset, development, or progression of osteoarthritis pain in anindividual, said method comprising administering an effective amount ofan antagonist of NGF (such as anti-NGF antagonist antibody) to theindividual.

In another aspect, the invention provides methods for improving physicalfunction in an individual having osteoarthritis, said method comprisingadministering an effective amount of an antagonist of NGF (such asanti-NGF antagonist antibody) to the individual.

In another aspect, the invention provides methods for improvingstiffness in an individual having osteoarthritis, said method comprisingadministering an effective amount of an antagonist of NGF (such asanti-NGF antagonist antibody) to the individual.

In some embodiments, the individual is a human. In some embodiments, fortreating osteoarthritis pain, dosing frequency of anti-NGF antagonistantibody is between once every week and once every 10 weeks, or lessfrequent.

In another aspect, the invention provides kits and compositionscomprising any one or more of the compositions described herein. Thesekits, generally in suitable packaging and provided with appropriateinstructions, are useful for any of the methods described herein.

The invention also provides any of the compositions and kits describedfor any use described herein whether in the context of use as medicamentand/or use for manufacture of a medicament.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: shows the amino acid sequence of the heavy chain variableregion of the E3 antibody (labeled “6” and “5+affinity maturation H3).The Chothia CDRs and Kabat CDRs are depicted by underlined text and boldand italicized text, respectively. FIG. 1A also shows the alignment ofthe following heavy chain variable region amino acid sequences; (2)VH4-59 human germline acceptor sequence (labeled “VH4-59” or “2”); (3)the acceptor sequences grafted with the extended CDRs of the mouseantibody 911 (labeled “CDR grafted” or “3”); (4) the CDR graftedacceptor sequences including the V71K substitution (labeled “3+oneframework mutation” or “4”); (5) the clone containing affinity maturedCDRs H1 and H2 (labeled “5” or “4+ affinity maturation H1, H2”); andantibody E3 (as described above).

FIG. 1B: shows the amino acid sequence of the light chain variableregion of the E3 antibody (labeled “5” or “4+ affinity maturation L3).The Chothia CDRs and Kabat CDRs are depicted by underlined text and boldand italicized text, respectively. FIG. 1B also shows the alignment ofthe following light chain variable region amino acid sequences: (2) 08human germline acceptor sequence (labeled “08” or “2”); (3) the acceptorsequences grafted with the extended CDRs of the mouse antibody 911(labeled “CDR grafted” or “3”); (4) the CDR grafted acceptor sequences(labeled “3+ affinity maturation L1, L2” or “4”); (5) the clonecontaining affinity matured CDRs L1 and L2 (labeled “5” or “4+ affinitymaturation L3”); and antibody E3 (as described above).

FIG. 2: shows a polynucleotide comprising a polynucleotide sequenceencoding the heavy chain variable region of antibody E3.

FIG. 3: shows a polynucleotide comprising a polynucleotide sequenceencoding the light chain variable region of antibody E3.

FIG. 4: is a graph depicting NGF-dependent survival of E13.5 neurons inthe presence of varying concentration of human and rat NGF. The X axiscorresponds to NGF concentration (ng/ml) and the Y axis corresponds tocounted neurons.

FIG. 5: is a graph comparing the NGF blocking effect of various Fabs inthe presence of either 0.04 ng/ml of human NGF (approximately 1.5 pM;shown in lower panel) or 0.4 ng/ml human NGF (approximately 15 pM; shownin upper panel). Survival of E13.5 mouse trigeminal neurons in variousconcentrations of Fab E3; murine 911 Fab; and Fab H19-L129 and Fab8L2-6D5 was assessed. The IC50 (in pM) was calculated for each Fab ateach NGF concentration, and is shown in Table 9. Fab E3 strongly blockedhuman NGF-dependent trigeminal neuron survival, with an IC50 ofapproximately 21 pM in the presence of 15 pM human NGF, and an IC50 ofapproximately 1.2 pM in the presence of 1.5 pM human NGF. Fabs 3C andH19-L129 also strongly blocked human NGF-dependent trigeminal neuronsurvival. In both panels, the X axis corresponds to antibodyconcentration (nM) and the Y axis corresponds to counted neurons. 1.5 pMof NGF was around the IC50, while 15 pM represented a saturatingconcentration of NGF.

FIG. 6: is a graph comparing the NGF blocking effect of various Fabs inthe presence of either 0.04 ng/ml of rat NGF (approximately 1.5 pM;shown in lower panel) or 0.4 ng/ml rat NGF (approximately 15 pM; shownin upper panel). Survival of E13.5 mouse trigeminal neurons in variousconcentrations of Fab E3; murine Fab 911; and Fab H19-L129 and 8L2-6D5was assessed as described above. The IC50 (in pM) was calculated foreach Fab at each NGF concentration, and is shown in Table 9. Fab E3strongly blocked human NGF-dependent trigeminal neuron survival, with anIC50 of approximately 31.6 pM in the presence of 15 pM rat NGF, and anIC50 of approximately 1.3 pM in the presence of 1.5 pM rat NGF. Fabs 3Cand H19-L129 also strongly blocked rat NGF-dependent trigeminal neuronsurvival. 1.5 pM of NGF was around the IC50, while 15 pM represented asaturating concentration of NGF. In both panels, the X axis correspondsto antibody concentration (nM) and the Y axis corresponds to countedneurons.

FIG. 7: is a graph depicting resting pain assessed 24 hours aftersurgery and showing that treatment with 0.02 mg/kg, 0.1 mg/kg, 0.6mg/kg, or 1 mg/kg of anti-NGF antibody E3 reduced pain. “*” indicates astatistically significant difference (p<0.5) from the negative control.

FIG. 8: is a graph depicting resting pain assessed 24 hours aftersurgery and showing that treatment with 0.5 mg/kg of anti-NGF antibodyE3 significantly (p<0.005) reduced resting pain when injected two hoursafter surgery.

FIG. 9: is a graph showing the results of BIAcore analysis of thebinding affinity to human NGF of mouse antibody 911 (Fab). Mouseantibody 911 bound NGF with a KD of 3.7 nM, koff of 8.4×10-5s-1 and konof 2.2×104 Ms-1.

FIG. 10: is a graph showing the results of BIAcore analysis of thebinding affinity to human NGF of antibody E3 (Fab) (referred to as “3EFab”). E3 bound human NGF with a KD of approximately 0.07 nM (and with akon of about 6.0×10⁵ M⁻¹s⁻¹, and a k_(off) of about 4.2×10⁻⁵ s⁻¹).

FIG. 11: is a graph depicting that antibody E3 blocks the interaction ofNGF with its receptors, trkA and p75, as assessed by percent bindingdetected between NGF and trkA (shown in black circles) and NGF and p75(shown as hollow squares). The X axis corresponds to concentration ofantibody 3E (Fab) and the Y axis corresponds to NGF binding (percentmaximum RU). Increased concentrations of Fab E3 blocked the interactionof NGF with both p75 and trkA, as shown by decreased signal (measured inRU). When antibody E3 (Fab) concentration equaled NGF concentration, noNGF binding was observed (as shown by a signal of zero).

FIG. 12: is a graph depicting the human NGF blocking ability of fullantibody E3 and Fab E3. Survival of E13.5 mouse trigeminal neurons inthe presence of human NGF and various concentrations of Fab E3 andantibody E3 was assessed. The X axis corresponds to NGF binding sites(nM) and the Y axis corresponds to normalized count of trigeminal (TG)neurons. Full antibody E3 and Fab 3E showed similar levels of inhibitionof NGF-dependent survival of trigeminal neurons when the concentrationof whole antibody and Fab were normalized to the number of NGF bindingsites (Fab has one binding site and whole antibody has two bindingsites).

FIG. 13: is a graph depicting the ability of various concentrations (20,4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.0 nM) of antibody E3 (solidtriangles; referred to as “3E”), antibody 911 (solid circles), and atrkA receptor immunoadhesin (shaded squares; referred as “trkA-Fc) toinhibit NGF-dependent survival of E13.5 trigeminal neurons in thepresence of 0.4 ng/ml human NGF (saturating conditions). The X axiscorresponds to concentration of antibody (nM) and the Y concentrationcorresponds to counted neurons. These results demonstrated that antibodyE3 blocked NGF significantly better than either mouse monoclonalanti-NGF antibody 911 or the trkA immunoadhesin.

FIG. 14: is a graph depicting that anti-NGF antagonist antibody E3(termed “3E in the figure”) or Fab 911 did not inhibit the neuronalsurvival promoted by NT3, NT4/5 and MSP, even at antibody concentrationsas high as 200 nM. The data represented mean percent survival after 48hours in culture (±standard error of mean, n=3 for each data point)relative to the survival observed in the positive control for eachexperiment (100% survival of trigeminal neurons grown in the presence ofsaturating NGF concentration). Various concentrations (20 nM, 2 nM, or0.2 nM) of E3 Fab (termed “3E” in the figure) and mouse antibody 911 Fabwere used in the presence of no added neurotrophin (termed “control”),400 pM NGF (termed “NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM) or 600 pMMSP (termed “MSP-600 pM).

FIG. 15: is a graph depicting that anti-NGF antagonist antibody E3 (Fabor full antibody) (termed “3E in the figure”) or mouse antibody 911 (Fabor full antibody) did not inhibit the neuronal survival promoted by NT3,NT4/5 and MSP, even at antibody concentrations as high as 200 nM Variousconcentrations (200 nM and 80 nM) of E3 Fab and full antibody and mouseantibody 911 full antibody and Fab were used in the presence of no addedneurotrophins (termed “no factor”), 400 pM NGF (termed “NGF-400 pM), 10nM NT3 (termed “NT3-10 nM) or 600 pM MSP (termed “MSP-600 pM).

FIG. 16: is a graph depicting that anti-NGF antagonist antibody E3 orFab E3 did not inhibit survival of E17 nodose neurons promoted by BDNF,NT4/5 or LIF. Mouse anti-NGF antagonist antibody 911 was also tested,and similar results were observed. Various concentrations (200 nM or 80nM) of full antibody E3 (termed “3E in the figure”), Fab E3, fullantibody 911, or Fab 911 were tested in the presence of no addedneurotrophins (termed “no factors”), 400 pM BDNF (termed “BDNF-400 pM),400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nM LIF (termed “LIF-2.5 nM).

FIG. 17: is a graph depicting that anti-NGF antagonist antibody E3 orFab E3 did not inhibit survival of E17 nodose neurons promoted by BDNF,NT4/5 or LIF. Various concentrations (200 nM, 20 nM, 2 nM) of Fab E3(termed “3E in the figure”), or Fab 911 were tested in the presence ofno added neurotrophins (termed “control”), 400 pM BDNF (termed “BDNF-400pM), 400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nM LIF (termed “LIP-2.5nM).

FIG. 18: is a graph demonstrating nociceptive response in arthritic rats(rheumatoid arthritis model) after administration of anti-NGF antibodies(E3 and 911) on D14 and D19. E3 (1 mg/kg, i.v. on day 14 and day 19),911 (10 mg/kg, i.v. on day 14 and day 19), or indo (indomethacin 3mg/kg, p.o. daily over 10 days) were administered to arthritic mice.Vocalization intensity values are expressed in mV as means±s.e.m.

FIG. 19: is a graph demonstrating effects of anti-NGF antibodies on bodyweight in arthritis in rats (rheumatoid arthritis model) afteradministration of anti-NGF antibodies on D14 and D19. E3 (1 mg/kg, i.v.on day 14 and day 19), 911 (10 mg/kg, i.v. on day 14 and day 19), orindo (indomethacin 3 mg/kg, p.o. daily over 10 days) were administeredto arthritic mice. Body weight values are expressed in grams asmean±s.e.m.

FIG. 20: is a graph demonstrating nociceptive response in arthritic rats(rheumatoid arthritis model) after administration of different doses ofanti-NGF antibody E3 (0.003 mg/kg, 0.03 mg/kg, 0.3 mg/kg, and 5 mg/kg)on D14 and D18. Vocalization intensity values are expressed in mV asmeans±s.e.m.

FIG. 21: is a graph demonstrating effects of anti-NGF antibody E3 onpercentage of weight on Day 14 (normalized to Day 14) in arthritic rats(rheumatoid arthritis model) after administration of different doses ofanti-NGF antibody E3 (0.03 mg/kg, 0.3 mg/kg, and 5 mg/kg) on D14 andD18.

FIG. 22: is a graph demonstrating effects of anti-NGF antibody E3 onweight loss in arthritic rats (rheumatoid arthritis model) afteradministration of different doses of anti-NGF antibody E3 (0.03 mg/kg,0.3 mg/kg, and 5 mg/kg) on D14 and D18. Body weight values werenormalized to Day 0.

FIG. 23A and FIG. 23B: depict the E3 heavy chain variable region aminoacid sequence (FIG. 23A) (SEQ ID NO: 1) and light chain variable regionamino acid sequence (FIG. 23B) (SEQ ID NO: 2), as numbered usingsequential numbering, Kabat numbering, and Chothia numbering.

FIG. 24 depicts the changes in mean daily pain intensity afteradministration of anti-NGF antibody E3 as compared to baseline on day 0.Y axis corresponds to reduction in mean daily pain intensity (VAS score)as compared to mean daily pain intensity on day 0. X axis corresponds todays after administration of anti-NGF antibody E3.

FIG. 25 depicts mean VAS score after administration of anti-NGF antibodyE3. “SE” refers to standard error.

FIG. 26 depicts percent maximal reduction in summed pain intensitydifference (SPID) from day 2 to day 14 and from day 2 to day 28 afteradministration of anti-NGF antibody E3.

FIG. 27 depicts least square mean (LSM) response for WOMAC, painsubscale, physical function subscale, and stiffness subscale from day 1to day 28 after administration of different doses (3 μg/kg, 10 μg/kg, 30μg/kg, 100 μg/kg, and 300 μg/kg) of anti-NGF antibody E3. “SE” refers tostandard error. X axes correspond to dose of anti-NGF antibody E3administered. “*” indicates P<0.05 as compared to baseline underDunnett's test.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides anti-NGF antagonist antibodiesthat bind NGF (such as human NGF) with high affinity. The inventionfurther provides antibodies and polypeptides derived from E3 that bindNGF, and methods of making and using these antibodies. In someembodiments, the invention provides a humanized antibody, E3, whichbinds to nerve growth factor (“NGF”), and methods of making and usingthis antibody. The invention also provides E3 polypeptides (includingantibodies) that bind NGF, and polynucleotides encoding E3 antibodyand/or polypeptide.

The invention disclosed herein also provides methods for preventingand/or treating rheumatoid arthritis pain in an individual byadministration of a therapeutically effective amount of an anti-NGFantagonist antibody.

The invention disclosed herein also provides methods for preventingand/or treating osteoarthritis pain in an individual by administrationof a therapeutically effective amount of an antagonist of NGF (such asan anti-NGF antagonist antibody).

The invention also provides methods for adjusting the affinity of anantibody and methods for characterizing a CDR region.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:Principles and Practice of Oncology (V. T. DeVita et al., eds., J. B.Lippincott Company, 1993).

Definitions

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv), mutants thereof, fusion proteins comprising an antibody portion,and any other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site. An antibody includes an antibodyof any class, such as IgG, IgA, or IgM (or sub-class thereof), and theantibody need not be of any particular class. Depending on the antibodyamino acid sequence of the constant domain of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known

“Fv” is an antibody fragment that contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy and one light chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy and one light chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a dimeric structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding specificity on thesurface of the VH-VL dimer. However, even a single variable domain (orhalf of a Fv comprising only 3 CDRs specific for an antigen) has theability to recognize and bind antigen, although generally at a loweraffinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge regions.

A “monoclonal antibody” refers to a homogeneous antibody populationwherein the monoclonal antibody is comprised of amino acids (naturallyoccurring and non-naturally occurring) that are involved in theselective binding of an antigen. A population of monoclonal antibodiesis highly specific, being directed against a single antigenic site. Theterm “monoclonal antibody” encompasses not only intact monoclonalantibodies and full-length monoclonal antibodies, but also fragmentsthereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain (ScFv), mutantsthereof, fusion proteins comprising an antibody portion, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site of the required specificity and the ability tobind to an antigen. It is not intended to be limited as regards to thesource of the antibody or the manner in which it is made (e.g., byhybridoma, phage selection, recombinant expression, transgenic animals,etc.).

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orhas been made using any of the techniques for making human antibodiesknown in the art or disclosed herein. This definition of a humanantibody includes antibodies comprising at least one human heavy chainpolypeptide or at least one human light chain polypeptide. One suchexample is an antibody comprising murine light chain and human heavychain polypeptides. Human antibodies can be produced using varioustechniques known in the art. In one embodiment, the human antibody isselected from a phage library, where that phage library expresses humanantibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314;Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter,1991, J Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,222:581). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. This approach is described in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.Alternatively, the human antibody may be prepared by immortalizing humanB lymphocytes that produce an antibody directed against a target antigen(such B lymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J.Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.

“Chimeric antibodies” refers to those antibodies wherein one portion ofeach of the amino acid sequences of heavy and light chains is homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular class, while the remaining segmentof the chains is homologous to corresponding sequences in another.Typically, in these chimeric antibodies, the variable region of bothlight and heavy chains mimics the variable regions of antibodies derivedfrom one species of mammals, while the constant portions are homologousto the sequences in antibodies derived from another. One clear advantageto such chimeric forms is that, for example, the variable regions canconveniently be derived from presently known sources using readilyavailable hybridomas or B cells from non human host organisms incombination with constant regions derived from, for example, human cellpreparations. While the variable region has the advantage of ease ofpreparation, and the specificity is not affected by its source, theconstant region being human, is less likely to elicit an immune responsefrom a human subject when the antibodies are injected than would theconstant region from a non-human source. However, the definition is notlimited to this particular example.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor; BCR),etc. Such effector functions generally require the Fc region to becombined with a binding domain (e.g. an antibody variable domain) andcan be assessed using various assays known in the art for evaluatingsuch antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

As used herein “antibody-dependent cell-mediated cytotoxicity” and“ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxiccells that express Fc receptors (FcRs) (e.g. natural killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., 1998, PNAS (USA),95:652-656.

As used herein, “Fc receptor” and “FcR” describe a receptor that bindsto the Fc region of an antibody. The preferred FcR is a native sequencehuman FcR. Moreover, a preferred FcR is one which binds an IgG antibody(a gamma receptor) and includes receptors of the FcγRI, FcγRII, andFcγRIII subclasses, including allelic variants and alternatively splicedforms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRII1B (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods,4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. “FcR”also includes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,117:587; and Kim et al., 1994, J. Immunol., 24:249).

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202:163 (1996), may beperformed.

As used herein, the terms “E3”, “3E”, and “antibody E3” are usedinterchangeably to refer to an antibody comprising the amino acidsequence of the heavy chain and light chain variable regions shown inFIGS. 1A (SEQ ID NO:1) and 1B (SEQ ID NO:2), respectively. The CDRportions of antibody E3 (including Chothia and Kabat CDRs) arediagrammatically depicted in FIGS. 1A and 1B. FIGS. 2 and 3 showpolynucleotides encoding heavy and light chains, respectively,comprising the heavy and light chain variable regions shown in FIGS. 1Aand 1 i, respectively. The generation and characterization of E3 isdescribed in the Examples. Different biological functions are associatedwith E3, including, but not limited to, ability to bind to NGF andinhibit NGF biological activity and/or downstream pathway(s) mediated byNGF signaling; and ability to inhibit NGF-dependent survival of mouseE13.5 trigeminal neurons. As discussed herein, antibodies of theinvention may have any one or more of these characteristics. In someembodiments, the term “E3” refers to immunoglobulin encoded by (a) apolynucleotide encoding E3 light chain that has a deposit number of ATCCNo. PTA-4893 or ATCC No. PTA-4894, and (b) a polynucleotide encoding E3heavy chain that has a deposit number of ATCC No. PTA-4895.

As used herein, “immunospecific” binding of antibodies refers to theantigen specific binding interaction that occurs between theantigen-combining site of an antibody and the specific antigenrecognized by that antibody (i.e., the antibody reacts with the proteinin an ELISA or other immunoassay, and does not react detectably withunrelated proteins).

An epitope that “specifically binds”, or “preferentially binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an NGF epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other NGF epitopes or non-NGFepitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon an antibody, the polypeptides can occur as single chains orassociated chains.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase. A polynucleotidemay comprise modified nucleotides, such as methylated nucleotides andtheir analogs. If present, modification to the nucleotide structure maybe imparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified after polymerization, such as byconjugation with a labeling component. Other types of modificationsinclude, for example, “caps”, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)and with charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.), those containing pendant moieties, such as, for example, proteins(e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine,etc.), those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupsmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,α-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Chothia et al. (1989) Nature342:877; Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). As usedherein, a CDR may refer to CDRs defined by either approach or by acombination of both approaches.

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination.

As used herein, the term “nerve growth factor” and “NGF” refers to nervegrowth factor and variants thereof that retain at least part of thebiological activity of NGF. As used herein, NGF includes all mammalianspecies of native sequence NGF, including human, canine, feline, equine,or bovine.

“NGF receptor” refers to a polypeptide that is bound by or activated byNGF. NGF receptors include the TrkA receptor and the p75 receptor of anymammalian species, including, but are not limited to, human, canine,feline, equine, primate, or bovine.

As used herein, an “anti-NGF antagonist antibody” (interchangeablytermed “anti-NGF antibody”) refers to an antibody which is able to bindto NGF and inhibit NGF biological activity and/or downstream pathway(s)mediated by NGF signaling. An anti-NGF antagonist antibody encompassesantibodies that block, antagonize, suppress or reduce (includingsignificantly) NGF biological activity, including downstream pathwaysmediated by NGF signaling, such as receptor binding and/or elicitationof a cellular response to NGF. For purpose of the present invention, itwill be explicitly understood that the term “anti-NGF antagonistantibody” encompass all the previously identified terms, titles, andfunctional states and characteristics whereby the NGF itself, an NGFbiological activity (including but not limited to its ability to abilityto mediate any aspect of post-surgical pain), or the consequences of thebiological activity, are substantially nullified, decreased, orneutralized in any meaningful degree. In some embodiments, an anti-NGFantagonist antibody binds NGF and prevent NGF dimerization and/orbinding to an NGF receptor (such as p75 and/or trkA). In otherembodiments, an anti-NGF antibody binds NGF and prevents trkA receptordimerization and/or trkA autophosphorylation. Examples of anti-NGFantagonist antibodies are provided herein.

“Biological activity” of NGF generally refers to the ability to bind NGFreceptors and/or activate NGF receptor signaling pathways. Withoutlimitation, a biological activity includes any one or more of thefollowing: the ability to bind an NGF receptor (such as p75 and/ortrkA); the ability to promote trkA receptor dimerization and/orautophosphorylation; the ability to activate an NGF receptor signalingpathway; the ability to promote cell differentiation, proliferation,survival, growth and other changes in cell physiology, including (in thecase of neurons, including peripheral and central neuron) change inneuronal morphology, synaptogenesis, synaptic function, neurotransmitterand/or neuropeptide release and regeneration following damage; theability to promote survival of mouse E13.5 trigeminal neurons; and theability to mediate pain, including post-surgical pain.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably at least90% pure, more preferably at least 95% pure, more preferably at least98% pure, more preferably at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improvement or alleviation of any aspect of pain,including acute, chronic, inflammatory, neuropathic, post-surgical pain,rheumatoid arthritis pain, or osteoarthritis pain. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, one or more of the following: including lessening severity,alleviation of one or more symptoms associated with pain including anyaspect of pain (such as shortening duration of pain, reduction of painsensitivity or sensation).

An “effective amount” of drug, compound, or pharmaceutical compositionis an amount sufficient to effect beneficial or desired resultsincluding clinical results such as alleviation or reduction in painsensation. An effective amount can be administered in one or moreadministrations. For purposes of this invention, an effective amount ofdrug, compound, or pharmaceutical composition is an amount sufficient totreat, ameliorate, reduce the intensity of and/or prevent pain,including post-surgical pain, rheumatoid arthritis pain, and/orosteoarthritis pain. In some embodiments, the “effective amount” mayreduce pain at rest (resting pain) or mechanically-induced pain(including pain following movement), or both, and it may be administeredbefore, during or after an incision, cut, tear or injury and/or before,during or after painful stimulus. As is understood in the clinicalcontext, an effective amount of a drug, compound, or pharmaceuticalcomposition may or may not be achieved in conjunction with another drug,compound, or pharmaceutical composition. Thus, an “effective amount” maybe considered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

“Reducing incidence” of pain means any of reducing severity (which caninclude reducing need for and/or amount of (e.g., exposure to) otherdrugs and/or therapies generally used for this conditions, including,for example, opiates), duration, and/or frequency (including, forexample, delaying or increasing time to post-surgical pain in anindividual). As is understood by those skilled in the art, individualsmay vary in terms of their response to treatment, and, as such, forexample, a “method of reducing incidence of rheumatoid arthritis pain orosteoarthritis pain in an individual” reflects administering theanti-NGF antagonist antibody based on a reasonable expectation that suchadministration may likely cause such a reduction in incidence in thatparticular individual.

“Ameliorating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means a lessening orimprovement of one or more symptoms of a pain as compared to notadministering an anti-NGF antagonist antibody. “Ameliorating” alsoincludes shortening or reduction in duration of a symptom.

“Palliating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means lessening theextent of one or more undesirable clinical manifestations ofpost-surgical pain in an individual or population of individuals treatedwith an anti-NGF antagonist antibody in accordance with the invention.

As used therein, “delaying” the development of pain means to defer,hinder, slow, retard, stabilize, and/or postpone progression of pain,such as post-surgical pain, rheumatoid arthritis pain, or osteoarthritispain. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individuals being treated. As is evidentto one skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developpain. A method that “delays” development of the symptom is a method thatreduces probability of developing the symptom in a given time frameand/or reduces extent of the symptoms in a given time frame, whencompared to not using the method. Such comparisons are typically basedon clinical studies, using a statistically significant number ofsubjects.

“Pain” as used herein refers to pain of any etiology, including acuteand chronic pain, and any pain with an inflammatory component. Examplesof pain include post-surgical pain, post-operative pain (includingdental pain), migraine, headache and trigeminal neuralgia, painassociated with burn, wound or kidney stone, pain associated with trauma(including traumatic head injury), neuropathic pain, pain associatedwith musculo-skeletal disorders such as rheumatoid arthritis,osteoarthritis, ankylosing spondylitis, sero-negative (non-rheumatoid)arthropathies, non-articular rheumatism and peri-articular disorders,and pain associated with cancer (including “break-through pain” and painassociated with terminal cancer), peripheral neuropathy andpost-herpetic neuralgia. Examples of pain with an inflammatory component(in addition to some of those described above) include rheumatic pain,pain associated with mucositis, and dysmenorrhea.

“Post-surgical pain” (interchangeably termed “post-incisional” or“post-traumatic pain”) refers to pain arising or resulting from anexternal trauma such as a cut, puncture, incision, tear, or wound intotissue of an individual (including that that arises from all surgicalprocedures, whether invasive or non-invasive). As used herein,post-surgical pain does not include pain that occurs (arises ororiginates) without an external physical trauma. In some embodiments,post-surgical pain is internal or external (including peripheral) pain,and the wound, cut, trauma, tear or incision may occur accidentally (aswith a traumatic wound) or deliberately (as with a surgical incision).As used herein, “pain” includes nociception and the sensation of pain,and pain can be assessed objectively and subjectively, using pain scoresand other methods well-known in the art. Post-surgical pain, as usedherein, includes allodynia (i.e., increased response to a normallynon-noxious stimulus) and hyperalgesia (i.e., increased response to anormally noxious or unpleasant stimulus), which can in turn, be thermalor mechanical (tactile) in nature. In some embodiments, the pain ischaracterized by thermal sensitivity, mechanical sensitivity and/orresting pain. In some embodiments, the post-surgical pain comprisesmechanically-induced pain or resting pain. In other embodiments, thepost-surgical pain comprises resting pain. The pain can be primary orsecondary pain, as is well-known in the art.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows), sport animals, pets (such as cats, dogs and horses), primates,mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline. Compositions comprising such carriers are formulated by wellknown conventional methods (see, for example, Remington's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990; and Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing, 2000).

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “Kd”, as used herein, is intended to refer to the dissociationconstant of an antibody-antigen interaction.

Antibody E3, E3-Derived Antibodies, Compositions, and Methods of Use E3Compositions, E3 Derived Compositions, and Methods of Making theCompositions

This invention encompasses compositions, including pharmaceuticalcompositions, comprising an E3 antibody or polypeptide; andpolynucleotides comprising sequences encoding an E3 antibody orpolypeptide. As used herein, compositions comprise one or moreantibodies or polypeptides (which may or may not be an antibody) thatbind to NGF, and/or one or more polynucleotides comprising sequencesencoding one or more antibodies or polypeptides that bind to NGF. Thesecompositions may further comprise suitable excipients, such aspharmaceutically acceptable excipients including buffers, which are wellknown in the art.

The invention also encompasses isolated antibody, polypeptide andpolynucleotide embodiments. The invention also encompasses substantiallypure antibody, polypeptide and polynucleotide embodiments.

The antibodies and polypeptides of the invention are characterized byany (one or more) of the following characteristics: (a) ability to bindto NGF; (b) ability to reduce and/or inhibit NGF biological activityand/or downstream pathway(s) mediated by NGF signaling; (c) ability toreduce and/or inhibit NGF-dependent survival of mouse E13.5 trigeminalneurons; (d) absence of any significant cross-reactivity to NT3, NT4/5,and/or BDNF; (e) ability to treat and/or prevent pain (includingpost-surgical pain); (f) ability to increase clearance of NGF; (g)ability to reduce or inhibit activation of trkA receptor, as detected,for example, using kinase receptor activation assay (KIRA) (see U.S.Pat. No. 6,027,927).

The binding properties of antibody E3, which binds human NGF with highaffinity and slow dissociation kinetics, compared with parent murineanti-NGF monoclonal antibody 911, are summarized below. E3 binds humanNGF with an approximately 50-fold higher binding affinity than parentmouse antibody 911.

antibody k_(D) K_(off) K_(on) 911 (Fab)  3.7 nM  9 × 10⁻⁵s⁻¹ 2.2 ×10⁴M⁻¹s⁻¹ E3 (Fab) 0.07 nM <4 × 10⁻⁵s⁻¹   6 × 10⁵M⁻¹s⁻¹

The E3 antibody and related antibodies also exhibit a strong capacity toantagonize human NGF, as assessed by in vitro assays (see Examples 2 and3). For example, antibody E3 antagonizes the NGF-dependent survival ofmouse E13 trigeminal neurons at an IC50 of about 21 pM in the presenceof 15 pM of human NGF, and about 1.2 pM in the presence of 1.5 pM ofhuman NGF.

Accordingly, in another aspect, the antibodies and polypeptides of theinvention are further identified and characterized by: (h) high affinitybinding to human NGF with low dissociation kinetics (in someembodiments, with a K_(D) of less than about 2 nM, and/or a koff ofslower than about 6×10-5 s-1) and/or (i) ability to inhibit (block)NGF-dependent survival of mouse E13.5 trigeminal neurons with an IC50 ofabout 100 pM or less at about 15 pM of NGF (in some embodiments, humanNGF) and/or an IC50 of about 20 pM or less at about 1.5 pM of NGF.

In some embodiments, the antibody binds human NGF, and does notsignificantly bind an NGF from another vertebrate species (in someembodiment, mammalian). In some embodiments, the antibody binds humanNGF as well as one or more NGF from another vertebrate species (in someembodiments, mammalian). In still other embodiments, the antibody bindsNGF and does not significantly cross-react with other neurotrophins(such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In someembodiments, the antibody binds NGF as well as at least one otherneurotrophin. In some embodiments, the antibody binds to a mammalianspecies of NGF, such as horse or dog, but does not significantly bind toNGF from anther mammalian species.

In some embodiments, the invention is an antibody comprising a lightchain that is encoded by a polynucleotide that is produced by a hostcell with a deposit number of ATCC No. PTA-4893 or ATCC No. PTA-4894. Inanother aspect, the invention is an antibody comprising a heavy chainthat is encoded by a polynucleotide that is produced by a host cell witha deposit number of ATCC No. PTA-4895. The present invention alsoencompasses various formulations of E3 and equivalent antibody fragments(e.g., Fab, Fab′, F(ab′)₂, Fv, Fc, etc.), single chain (ScFv), mutantsthereof, fusion proteins comprising an antibody portion, and any othermodified configuration of E3 that comprises an antigen (NGF) recognitionsite of the required specificity. The equivalent antibodies of E3,including antibody and polypeptide fragments (which may or may not beantibodies) of E3, and polypeptides comprising polypeptide fragments ofE3 are identified and characterized by any (one or more) of the criteriadescribed above.

Accordingly, the invention provides any of the following, orcompositions (including pharmaceutical compositions) comprising any ofthe following: (a) antibody E3; (b) a fragment or a region of theantibody E3; (c) a light chain of the antibody E3 as shown in FIG. 1B;(c) a heavy chain of the antibody E3 as shown in FIG. 1A; (d) one ormore variable region(s) from a light chain and/or a heavy chain of theantibody E3; (e) one or more CDR(s) (one, two, three, four, five or sixCDRs) of antibody E3 shown in FIGS. 1A and 1B; (f) CDR H3 from the heavychain of antibody E3 shown in FIG. 1A; (g) CDR L3 from the light chainof antibody E3 shown in FIG. 1B; (h) three CDRs from the light chain ofantibody E3 shown in FIG. 1B; (i) three CDRs from the heavy chain ofantibody E3 shown in FIG. 1A; (j) three CDRs from the light chain andthree CDRs from the heavy chain, of antibody E3 shown in FIGS. 1A and1B; and (k) an antibody comprising any one of (b) through (j). As isevident from the description herein, specifically excluded from theinvention are polypeptide embodiments consisting of the identical aminoacid sequence to an amino acid sequence of mouse monoclonal antibody,911. The extended CDR sequences of Mab 911 are shown in FIGS. 1A and 1i, and in SEQ ID NOS:9-14.

The CDR portions of antibody E3 (including Chothia and Kabat CDRs) arediagrammatically depicted in FIGS. 1A and 1B, and consist of thefollowing amino acid sequences: (a) heavy chain CDR 1 (“CDR H1”)GFSLIGYDLN (SEQ ID NO:3); (b) heavy chain CDR 2 (“CDR H2”)IIWGDGTTDYNSAVKS (SEQ ID NO:4); (c) heavy chain CDR 3 (“CDR H3”)GGYWYATSYYFDY (SEQ ID NO:5); (d) light chain CDR 1 (“CDR L1”)RASQSISNNLN (SEQ ID NO:6); (e) light chain CDR 2 (“CDR L2”) YTSRFHS (SEQID NO:7); and (f) light chain CDR 3 (“CDR L3”) QQEHTLPYT (SEQ ID NO:8).Determination of CDR regions is well within the skill of the art. It isunderstood that in some embodiments, CDRs can be a combination of theKabat and Chothia CDR (also termed “combined CDRs” or “extended CDRs”).In some embodiments, the CDRs comprise the Kabat CDR. In otherembodiments, the CDRs are the Chothia CDR.

In some embodiments, the invention provides an antibody which comprisesat least one CDR that is substantially homologous to at least one CDR,at least two, at least three, at least four, at least 5 CDRs of E3 (or,in some embodiments substantially homologous to all 6 CDRs of E3, orderived from E3). Other embodiments include antibodies which have atleast two, three, four, five, or six CDR(s) that are substantiallyhomologous to at least two, three, four, five or six CDRs of E3 orderived from E3. It is understood that, for purposes of this invention,binding specificity and/or overall activity (which may be in terms oftreating and/or preventing pain or inhibiting NGF-dependent survival ofE13.5 mouse trigeminal neurons) is generally retained, although theextent of activity may vary compared to E3 (may be greater or lesser).

The invention also provides a polypeptide (which may or may not be anantibody) which comprises an amino acid sequence of E3 (shown in FIGS.1A and 1B) that has any of the following: at least 5 contiguous aminoacids, at least 8 contiguous amino acids, at least about 10 contiguousamino acids, at least about 15 contiguous amino acids, at least about 20contiguous amino acids, at least about 25 contiguous amino acids, atleast about 30 contiguous amino acids of a sequence of E3, wherein atleast 3 of the amino acids are from a variable region of E3, with theunderstanding that embodiments that consist of the identical amino acidsequence to an amino acid sequence of mouse monoclonal antibody, 911,are specifically excluded. The extended CDR sequences of Mab 911 areshown in FIGS. 1A and 1 i, and in SEQ ID NOS:9-14. In one embodiment,the variable region is from a light chain of E3. In another embodiment,the variable region is from a heavy chain of E3. In another embodiment,the 5 (or more) contiguous amino acids are from a complementaritydetermining region (CDR) of E3 shown in FIGS. 1A and 1B.

In another embodiment, the invention provides a polypeptide whichcomprises an amino acid sequence of E3 that has any of the following: atleast 5 contiguous amino acids, at least 8 contiguous amino acids, atleast about 10 contiguous amino acids, at least about 15 contiguousamino acids, at least about 20 contiguous amino acids, at least about 25contiguous amino acids, at least about 30 contiguous amino acids of asequence of E3, wherein the E3 sequence comprises any one or more of:amino acid residue L29 of CDRH1, I50 of CDRH2, W101 of CDRH3, and/orA103 of CDRH3; and/or amino acid residue S28 of CDRL1, N32 of CDRL1, T51of CDRL2, 91E of CDRL3 and/or H92 of CDRL3, with the understanding thatembodiments that consist of the identical amino acid sequence to anamino acid sequence of mouse monoclonal antibody, 911, are specificallyexcluded.

As is evident, throughout this disclosure, a sequential amino acidnumbering scheme is used to refer to amino acid residues in the variableregions (that is, the amino acid residues in each variable region arenumbered in sequence). As is well known in the art, the Kabat and/orChothia numbering systems are useful when comparing two antibodies orpolypeptides, such as an E3 antibody and an E3 variant (or polypeptidesuspected of being an E3 variant). It is well understood in the art howto convert sequential numbering to Chothia and/or Kabat numbering, ifdesired, for example, for use in making comparisons between E3 andanother polypeptide. FIG. 23A and FIG. 23B depict the E3 variableregions numbered using sequential, Chothia and Kabat numbering. Inaddition, to facilitate comparison, generally it is understood thatframework residues generally, but not always, have approximately thesame number of residues. However, the CDRs may vary in size (i.e., it ispossible to have insertions and/or deletions of one or more amino acidresidues). When comparing an E3 antibody and a candidate E3 variant (forexample, in the case of a CDR region from a candidate sequence which islonger in the sequence in antibody E3 to which is aligned), one mayfollow the following steps (though other methods are known in the art).The candidate antibody sequence is aligned with E3 antibody heavy chainand light chain variable regions. Alignment may be done by hand, or bycomputer using commonly accepted computer programs. Alignment may befacilitated by using some amino acid residues which are common to mostFab sequences. For example, the light and heavy chains each typicallyhave two cysteines, which are often found at a conserved position. It isunderstood that the amino acid sequence of a candidate variant antibodymay be longer (i.e. have inserted amino acid residues) or shorter (havedeleted amino acid residues). Suffixes may be added to the residuenumber to indicate the insertion of additional residues, e.g., residue34 abc. For candidate sequences which, for example, align with a E3sequence for, e.g., residues 33 and 35, but have no residue between themto align with residue 35, the residue 35 is simply not assigned to aresidue. In another approach, it is generally well known that comparisonmay be made between structural equivalent (e.g., same position in theantigen-antibody complex) amino acids when comparing CDRs of differentlengths. For example, the Chothia numbering (Al-Lazikani et al, supra)generally (but not in all cases), places insertions and deletions at thestructurally correct positions. Structural equivalence may also bededuced or demonstrated using X-ray crystallography or double mutantcycle analysis (see Pons et al. (1999) Prot. Sci. 8:958-968).

The binding affinity of an anti-NGF antibody to NGF (such as hNGF) canbe about 0.10 to about 0.80 nM, about 0.15 to about 0.75 nM and about0.18 to about 0.72 nM. In some embodiments, the binding affinity isabout 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, about 40pM, or greater than about 40 pM. In one embodiment, the binding affinityis between about 2 pM and 22 pM. In other embodiments, the bindingaffinity is less than about 10 nM, about 5 nM, about 4 nM, about 3.5 nM,about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about400 pM, about 300 pM, about 200 pM, about 150 pM, about 100 pM, about 90pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM,about 30 pM, about 10 pM. In some embodiments, the binding affinity isabout 10 nM. In other embodiments, the binding affinity is less thanabout 10 nM. In other embodiments, the binding affinity is about 0.1 nMor about 0.07 nM. In other embodiments, the binding affinity is lessthan about 0.1 nM or less than about 0.07 nM. In other embodiments, thebinding affinity is any of about 10 nM, about 5 nM, about 4 nM, about3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM,about 900 pM, about 800 pM, bout 700 pM, about 600 pM, about 500 pM,about 400 pM, about 300 pM, about 200 pM, about 150 pM, about 100 pM,about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about40 pM, about 30 pM, about 10 pM to any of about 2 pM, about 5 pM, about10 pM, about 15 pM, about 20 pM, or about 40 pM. In some embodiments,the binding affinity is any of about 10 nM, about 5 nM, about 4 nM,about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about1 nM, about 900 pM, about 800 pM, bout 700 pM, about 600 pM, about 500pM, about 400 pM, about 300 pM, about 200 pM, about 150 pM, about 100pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM,about 40 pM, about 30 pM, about 10 pM. In still other embodiments, thebinding affinity is about 2 pM, about 5 pM, about 10 pM, about 15 pM,about 20 pM, about 40 pM, or greater than about 40 pM.

The binding affinity of the antibody to NGF can be determined usingmethods well known in the art. One way of determining binding affinityof antibodies to NGF is by measuring affinity of monofunctional Fabfragments of the antibody, as described in the Examples. To obtainmonofunctional Fab fragments, an antibody (for example, IgG) can becleaved with papain or expressed recombinantly. The affinity of ananti-NGF Fab fragment of an antibody can be determined by surfaceplasmon resonance (BIAcore3000™ surface plasmon resonance (SPR) system,BIAcore, INC, Piscataway N.J.), as described in the Examples. Thisprotocol is suitable for use in determining binding affinity of anantibody to NGF of any species, including human NGF, NGF of anothervertebrate (in some embodiments, mammalian) (such as mouse NGF, rat NGF,primate NGF), as well as for use with other neurotrophins, such as therelated neurotrophins NT3, NT4/5, and/or BDNF.

In some embodiments, the antibodies or peptides of the invention mayinhibit (reduce, and/or block) human NGF-dependent survival of mouseE13.5 trigeminal neurons with an IC50 (in the presence of about 15 pM ofNGF) of about any of 200 pM, 150 pM, 100 pM, 80 pM, 60 pM, 40 pM, 20 pM,10 pM, or less. In some embodiments, the antibodies or peptides of theinvention may inhibit (reduce, and/or block) human NGF-dependentsurvival of mouse E13.5 trigeminal neurons with an IC50 (in the presenceof about 1.5 pM of NGF) of about any of 50 pM, 40 pM, 30 pM, 10 pM, 20pM, 10 pM, 5 pM, 2 pM, 1 pM, or less. In some embodiments, theantibodies or peptides of the invention may inhibit (reduce, and/orblock) rat NGF-dependent survival of mouse E13.5 trigeminal neurons withan IC50 (in the presence of about 15 pM of NGF) of about any of 150 pM,125 pM, 100 pM, 80 pM, 60 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or less.In some embodiments, the antibodies or peptides of the invention mayinhibit (reduce, and/or block) rat NGF-dependent survival of mouse E13.5trigeminal neurons with an IC50 (in the presence of about 1.5 pM of NGF)of about any of 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 5 pM, 4 pM, 3 pM, 2pM, 1 pM, or less. Methods for measurement of the NGF-dependent survivalof mouse E13 trigeminal neurons are known in the art, and described,e.g., in Example 2.

The invention also provides methods of making any of these antibodies orpolypeptides. The antibodies of this invention can be made by proceduresknown in the art, some of which are illustrated in the Examples. Thepolypeptides can be produced by proteolytic or other degradation of theantibodies, by recombinant methods (i.e., single or fusion polypeptides)as described above or by chemical synthesis. Polypeptides of theantibodies, especially shorter polypeptides up to about 50 amino acids,are conveniently made by chemical synthesis. Methods of chemicalsynthesis are known in the art and are commercially available. Forexample, a E3 antibody could be produced by an automated polypeptidesynthesizer employing the solid phase method. See also, U.S. Pat. Nos.5,807,715; 4,816,567; and 6,331,415. Chimeric or hybrid antibodies alsomay be prepared in vitro using known methods of synthetic proteinchemistry, including those involving cross-linking agents. For example,immunotoxins may be constructed using a disulfide exchange reaction orby forming a thioether bond. Examples of suitable reagents for thispurpose include iminothiolate and methyl-4-mercaptobutyrimidate.

In another alternative, the antibodies can be made recombinantly usingprocedures that are well known in the art. In one embodiment, apolynucleotide comprising a sequence encoding the variable and lightchain regions of antibody E3 (shown in FIGS. 1A and 1B) is cloned into avector for expression or propagation in a host cell (e.g., CHO cells).In another embodiment, the polynucleotide sequences shown in FIGS. 2 and3 are cloned into one or more vectors for expression or propagation. Thesequence encoding the antibody of interest may be maintained in a vectorin a host cell and the host cell can then be expanded and frozen forfuture use. Vectors (including expression vectors) and host cells arefurther described herein. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters et al. (2001) Vaccine 19:2756; Lonberg, N. and D. Huszar (1995)Int. Rev. Immunol 13:65; and Pollock et al. (1999) J Immunol Methods231:147. Methods for making derivatives of antibodies, e.g., humanized,single chain, etc. are known in the art.

The invention also encompasses single chain variable region fragments(“scFv”) of antibodies of this invention, such as E3. Single chainvariable region fragments are made by linking light and/or heavy chainvariable regions by using a short linking peptide. Bird et al. (1988)Science 242:423-426. An example of a linking peptide is (GGGGS)3 (SEQ IDNO:15), which bridges approximately 3.5 nm between the carboxy terminusof one variable region and the amino terminus of the other variableregion. Linkers of other sequences have been designed and used (Bird etal. (1988)). Linkers can in turn be modified for additional functions,such as attachment of drugs or attachment to solid supports. The singlechain variants can be produced either recombinantly or synthetically.For synthetic production of scFv, an automated synthesizer can be used.For recombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

The antibody may be a bispecific antibody, a monoclonal antibody thathas binding specificities for at least two different antigens. Abispecific antibody can be prepared using the antibodies disclosedherein. Methods for making bispecific antibodies are known in the art(see, e.g., Suresh et al., 1986, Methods in Enzymology 121:210).Traditionally, the recombinant production of bispecific antibodies wasbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, with the two heavy chains having different specificities(Millstein and Cuello, 1983, Nature 305, 537-539).

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690, published Mar. 3, 1994.

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT applicationpublication Nos. WO 91/00360 and WO 92/200373; EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents and techniques arewell known in the art, and are described in U.S. Pat. No. 4,676,980.

The antibody may be a humanized antibody, for example, as known in theart, and as described herein.

Antibodies may be modified as described in PCT Publication No. WO99/58572, published Nov. 18, 1999. These antibodies comprise, inaddition to a binding domain directed at the target molecule, aneffector domain having an amino acid sequence substantially homologousto all or part of a constant domain of a human immunoglobulin heavychain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. Preferably, the effector domainis capable of specifically binding FcRn and/or FcγRIIb. These aretypically based on chimeric domains derived from two or more humanimmunoglobulin heavy chain CH2 domains. Antibodies modified in thismanner are preferred for use in chronic antibody therapy, to avoidinflammatory and other adverse reactions to conventional antibodytherapy.

The invention encompasses modifications to antibody E3, includingfunctionally equivalent antibodies which do not significantly affecttheir properties and variants which have enhanced or decreased activity.Modification of polypeptides is routine practice in the art and isfurther exemplified in the Examples. Examples of modified polypeptidesinclude polypeptides with substitutions (including conservativesubstitutions) of amino acid residues, one or more deletions oradditions of amino acids which do not significantly deleteriously changethe functional activity, or use of chemical analogs.

A polypeptide “variant,” as used herein, is a polypeptide that differsfrom a native protein in one or more substitutions, deletions, additionsand/or insertions, such that the immunoreactivity of the polypeptide isnot substantially diminished. In other words, the ability of a variantto specifically bind antigen may be enhanced or unchanged, relative tothe native protein, or may be diminished by less than 50%, andpreferably less than 20%, relative to the native protein. Polypeptidevariants preferably exhibit at least about 80%, more preferably at leastabout 90% and most preferably at least about 95% identity (determined asdescribed herein) to the identified polypeptides.

Amino acid sequence variants of the antibodies may be prepared byintroducing appropriate nucleotide changes into the antibody DNA, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of SEQ ID NO:1 or 2 described herein. Anycombination of deletion, insertion, and substitution is made to arriveat the final construct, provided that the final construct possesses thedesired characteristics. The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis or modification iscalled “alanine scanning mutagenesis,” and is described by Cunninghamand Wells, 1989, Science, 244:1081-1085. A residue or group of targetresidues is identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity. Library scanningmutagenesis, as described herein, may also be used to identify locationsin an antibody that are suitable for mutagenesis or modification.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the serum half-life of the antibody.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 1 under the heading of“conservative substitutions”. If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Amino Acid Substitutions Original Conservative ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilic: Cys, Ser, Thr;

(3) Acidic: Asp, Glu;

(4) Basic: Asn, Gin, His, Lys, Arg;

(5) Residues that influence chain orientation: Gly, Pro; and

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity and/orspecificity. In some embodiment, no more than one to five conservativeamino acid substitutions are made within a CDR domain. In otherembodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR3 domain. In still other embodiments,the CDR domain is CDRH3 and/or CDR L3.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, MatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example usingendoglycosidase H (Endo H). In addition, the recombinant host cell canbe genetically engineered to be defective in processing certain types ofpolysaccharides. These and similar techniques are well known in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified E3 polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572, published Nov. 18,1999. These antibodies comprise, in addition to a binding domaindirected at the target molecule, an effector domain having an amino acidsequence substantially homologous to all or part of a constant domain ofa human immunoglobulin heavy chain. These antibodies are capable ofbinding the target molecule without triggering significant complementdependent lysis, or cell-mediated destruction of the target. In someembodiments, the effector domain is capable of specifically binding FcRnand/or FcγRIIb. These are typically based on chimeric domains derivedfrom two or more human immunoglobulin heavy chain CH2 domains.Antibodies modified in this manner are particularly suitable for use inchronic antibody therapy, to avoid inflammatory and other adversereactions to conventional antibody therapy.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies (such as E3) or polypeptides ofthis invention. In one embodiment, a fusion polypeptide is provided thatcomprises at least 10 contiguous amino acids of the variable light chainregion shown in FIG. 1B and/or at least 10 amino acids of the variableheavy chain region shown in FIG. 1A. In another embodiment, the fusionpolypeptide comprises a light chain variable region and/or a heavy chainvariable region of E3, as shown in FIGS. 1A and 1B. In anotherembodiment, the fusion polypeptide comprises one or more CDR(s) of E3.In still other embodiments, the fusion polypeptide comprises CDR H3and/or CDR L3 of antibody E3. In another embodiment, the fusionpolypeptide comprises any one or more of: amino acid residue L29 ofCDRH1, 150 of CDRH2, W101 of CDRH3, and/or A103 of CDRH3; and/or aminoacid residue S28 of CDRL1, N32 of CDRL1, T51 of CDRL2, 91E of CDRL3and/or H92 of CDRL3. For purposes of this invention, a E3 fusion proteincontains one or more E3 antibodies and another amino acid sequence towhich it is not attached in the native molecule, for example, aheterologous sequence or a homologous sequence from another region.Exemplary heterologous sequences include, but are not limited to a “tag”such as a FLAG tag or a 6His tag. Tags are well known in the art.

A E3 fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the E3 fusionproteins of this invention are made by preparing an expressing apolynucleotide encoding them using recombinant methods described herein,although they may also be prepared by other means known in the art,including, for example, chemical synthesis.

This invention also provides compositions comprising E3 antibodies orpolypeptides conjugated (for example, linked) to an agent thatfacilitate coupling to a solid support (such as biotin or avidin). Forsimplicity, reference will be made generally to E3 or antibodies withthe understanding that these methods apply to any of the NGF bindingembodiments described herein. Conjugation generally refers to linkingthese components as described herein. The linking (which is generallyfixing these components in proximate association at least foradministration) can be achieved in any number of ways. For example, adirect reaction between an agent and an antibody is possible when eachpossesses a substituent capable of reacting with the other. For example,a nucleophilic group, such as an amino or sulfhydryl group, on one maybe capable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

An antibody or polypeptide of this invention may be linked to a labelingagent (alternatively termed “label”) such as a fluorescent molecule, aradioactive molecule or any others labels known in the art. Labels areknown in the art which generally provide (either directly or indirectly)a signal. Accordingly, the invention includes labeled antibodies andpolypeptides.

The ability of the antibodies and polypeptides of this invention, suchas binding NGF; reducing or inhibiting a NGF biological activity;reducing and/or blocking NGF-induced survival of E13.5 mouse trigeminalneurons, may be tested using methods known in the art, some of which aredescribed in the Examples.

The invention also provides compositions (including pharmaceuticalcompositions) and kits comprising antibody E3, and, as this disclosuremakes clear, any or all of the antibodies and/or polypeptides describedherein.

Polynucleotides, Vectors and Host Cells

The invention also provides isolated polynucleotides encoding theantibodies and polypeptides of the invention (including an antibodycomprising the polypeptide sequences of the light chain and heavy chainvariable regions shown in FIGS. 1A and 1), and vectors and host cellscomprising the polynucleotide.

Accordingly, the invention provides polynucleotides (or compositions,including pharmaceutical compositions), comprising polynucleotidesencoding any of the following: (a) antibody E3; (b) a fragment or aregion of the antibody E3; (c) a light chain of the antibody E3 as shownin FIG. 1B; (d) a heavy chain of the antibody E3 as shown in FIG. 1A;(e) one or more variable region(s) from a light chain and/or a heavychain of the antibody E3; (f) one or more CDR(s) (one, two, three, four,five or six CDRs) of antibody E3 shown in FIGS. 1A and 1B; (g) CDR H3from the heavy chain of antibody E3 shown in FIG. 1A; (h) CDR L3 fromthe light chain of antibody E3 shown in FIG. 1B; (i) three CDRs from thelight chain of antibody E3 shown in FIG. 1B; ( ) three CDRs from theheavy chain of antibody E3 shown in FIG. 1A; (k) three CDRs from thelight chain and three CDRs from the heavy chain, of antibody E3 shown inFIGS. 1A and 1B; or (1) an antibody comprising any of (b) to (k). Insome embodiments, the polynucleotide comprises either or both of thepolynucleotide(s) shown in FIGS. 2 and 3.

In another aspect, the invention is an isolated polynucleotide thatencodes for an E3 light chain with a deposit number of ATCC No. PTA-4893or ATCC No. PTA-4894. In another aspect, the invention is an isolatedpolynucleotide that encodes for an E3 heavy chain with a deposit numberof ATCC No. PTA-4895. In yet another aspect, the invention is anisolated polynucleotide comprising (a) a variable region encoded in thepolynucleotide with a deposit number of ATCC No. PTA-4894 and (b) avariable region encoded in the polynucleotide with a deposit number ofATCC No. PTA-4895. In another aspect, the invention is an isolatedpolynucleotide comprising (a) one or more CDR encoded in thepolynucleotide with a deposit number of ATCC No. PTA-4894; and/or (b)one or more CDR encoded in the polynucleotide with a deposit number ofATCC No. PTA-4895.

In another aspect, the invention provides polynucleotides encoding anyof the antibodies (including antibody fragments) and polypeptidesdescribed herein.

Polynucleotides can be Made by Procedures Known in the Art

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the polynucleotides ofthe invention. In some embodiments, the composition comprises anexpression vector comprising a polynucleotide encoding the E3 antibodyas described herein. In other embodiment, the composition comprises anexpression vector comprising a polynucleotide encoding any of theantibodies or polypeptides described herein. In still other embodiments,the composition comprises either or both of the polynucleotides shown inFIGS. 2 and 3. Expression vectors, and administration of polynucleotidecompositions are further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably at least about 80% identity and most preferably at leastabout 90% identity to a polynucleotide sequence that encodes a nativeantibody or a portion thereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or10 to 12 percent, as compared to the reference sequences (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al. (1989).

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., (1989), for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtilis) and yeast (such as S. cerevisae, S. pombe; or K.lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably 10 fold higher, even morepreferably 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to NGF is effected by animmunoassay or FACS. A cell overexpressing the antibody or protein ofinterest can be identified.

Methods Using E3 and E3 Derived Antibodies

Antibody E3 which binds NGF may be used to identify or detect thepresence or absence of NGF. For simplicity, reference will be madegenerally to E3 or antibodies with the understanding that these methodsapply to any of the NGF binding embodiments (such as polypeptides)described herein. Detection generally involves contacting a biologicalsample with an antibody described herein that binds to NGF and theformation of a complex between NGF and an antibody (e.g., E3) whichbinds specifically to NGF. The formation of such a complex can be invitro or in vivo. The term “detection” as used herein includesqualitative and/or quantitative detection (measuring levels) with orwithout reference to a control.

Any of a variety of known methods can be used for detection, including,but not limited to, immunoassay, using antibody that binds thepolypeptide, e.g. by enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and the like; and functional assay for theencoded polypeptide, e.g. binding activity or enzymatic assay. In someembodiments, the antibody is detectably labeled.

Diagnostic Uses of the E3 and Derivatives

Antibodies and polypeptides of the invention can be used in thedetection, diagnosis and monitoring of a disease, condition, or disorderassociated with altered or aberrant NGF expression (in some embodiments,increased or decreased NGF expression (relative to a normal sample),and/or inappropriate expression, such as presence of expression intissue(s) and/or cell(s) that normally lack NGF expression, or absenceof NGF expression in tissue(s) or cell(s) that normally possess NGFexpression). The antibodies and polypeptides of the invention arefurther useful for detection of NGF expression, for example, in adisease associated with altered or aberrant sensitivity orresponsiveness to NGF. In some embodiments, NGF expression is detectedin a sample from an individual suspected of having a disease, disorderfeaturing or associated with an altered or aberrant sensitivity orresponsiveness to NGF expression (e.g., a cancer in which NGF promotesgrowth and/or metastasis).

Thus, in some embodiments, the invention provides methods comprisingcontacting a specimen (sample) of an individual suspected of havingaltered or aberrant NGF expression with an antibody or polypeptide ofthe invention and determining whether the level of NGF differs from thatof a control or comparison specimen. In some embodiments, the individualhas a cardiac arrhythmia, Alzheimer's disease, and/or autonomicdysfunction.

In other embodiments, the invention provides methods comprisescontacting a specimen (sample) of an individual and determining level ofNGF expression. In some embodiments, the individual is suspected ofhaving a disease, disorder featuring or associated with an altered oraberrant sensitivity or responsiveness to NGF expression. In someembodiments, the individual has small cell lung cancer, breast cancer,pancreatic cancer, prostate cancer, ovarian carcinoma, hepatocellularcarcinoma, or melanoma.

For diagnostic applications, the antibody typically will be labeled witha detectable moiety including but not limited to radioisotopes,fluorescent labels, and various enzyme-substrate labels. Methods ofconjugating labels to an antibody are known in the art. In otherembodiments of the invention, antibodies of the invention need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the antibodies of the invention.

The antibodies of the present invention may be employed in any knownassay method, such competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The antibodies may also be used for in vivo diagnostic assays, such asin vivo imaging. Generally, the antibody is labeled with a radionuclide(such as 111In, 99Tc, 14C, 131I, 125I, or 3H) so that the cells ortissue of interest can be localized using immunoscintiography.

The antibody may also be used as staining reagent in pathology,following techniques well known in the art.

Methods of Using E3 and Derivatives for Therapeutic Purposes

Antibody E3 is useful for reducing and/or blocking the biologicalactivity of NGF. This antagonistic activity is believed to be useful inthe treatment of pathological conditions associated with endogenous NGFproduction, such as pain. Generally, in these embodiments an effectiveamount is administered to an individual. Accordingly, in one aspect, theinvention provides a method of antagonizing human NGF biologicalactivity using any of the polypeptides (including antibodies such asantibody E3) disclosed herein. In one embodiment, the method comprisescontacting human nerve growth factor with any of the polypeptides(including antibody E3) described herein, whereby human nerve growthfactor activity is antagonized, reduced, blocked, or suppressed. In yetanother embodiment, an individual with pain (such as post-surgical pain,or rheumatoid arthritis pain) is given treatment with E3.

For simplicity, reference will be made generally to E3 or antibody withthe understanding that these methods apply to any of the E3 variantantibodies and polypeptides described herein.

Various formulations of E3 or fragments of E3 (e.g., Fab, Fab′, F(ab′)₂,Fv, Fc, etc.), such as single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, and any other modifiedconfiguration of E3 that comprises an antigen NGF recognition site ofthe required specificity, may be used for administration. In someembodiments, E3 antibodies or various formulations of E3 thereof may beadministered neat. In other embodiments, E3 or various formulations ofE3 (including any composition embodiment described herein) thereof and apharmaceutically acceptable excipient are administered, and may be invarious formulations. Pharmaceutically acceptable excipients are knownin the art, and are relatively inert substances that facilitateadministration of a pharmacologically effective substance. For example,an excipient can give form or consistency, or act as a diluent. Suitableexcipients include but are not limited to stabilizing agents, wettingand emulsifying agents, salts for varying osmolarity, encapsulatingagents, buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.), although other forms of administration (e.g.,oral, mucosal, via inhalation, sublingually, etc) can be also used.Accordingly, E3 antibody and equivalents thereof are preferably combinedwith pharmaceutically acceptable vehicles such as saline, Ringer'ssolution, dextrose solution, and the like. The particular dosageregimen, i.e., dose, timing and repetition, will depend on theparticular individual and that individual's medical history. Generally,any of the following doses may be used: a dose of at least about 50mg/kg body weight; at least about 10 mg/kg body weight; at least about 3mg/kg body weight; at least about 1 mg/kg body weight; at least about750 μg/kg body weight; at least about 500 μg/kg body weight; at leastabout 250 ug/kg body weight; at least about 100 μg/kg body weight; atleast about 50 μg/kg body weight; at least about 10 ug/kg body weight;at least about 1 μg/kg body weight, or less, is administered. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. An exemplary dosing regimen comprisesadministering an initial dose of about 2 mg/kg, followed by a weeklymaintenance dose of about 1 mg/kg of the anti-NGF antibody, or followedby a maintenance dose of about 1 mg/kg every other week. However, otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve. Empiricalconsiderations, such as the half-life, generally will contribute todetermination of the dosage. The progress of this therapy is easilymonitored by conventional techniques and assays.

In some individuals, more than one dose may be required. Frequency ofadministration may be determined and adjusted over the course oftherapy. For example, frequency of administration may be determined oradjusted based on the type and severity of the pain to be treated,whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, and the discretion of the attending physician. Typicallythe clinician will administer an anti-NGF antagonist antibody (such asE3), until a dosage is reached that achieves the desired result. In somecases, sustained continuous release formulations of E3 antibodies may beappropriate. Various formulations and devices for achieving sustainedrelease are known in the art.

In one embodiment, dosages for E3 antibodies (or polypeptides) may bedetermined empirically in individuals who have been given one or moreadministration(s). Individuals are given incremental dosages of E3. Toassess efficacy of E3 or other equivalent antibody, markers of thedisease symptoms (such as pain) can be monitored.

Administration of an antibody (such as E3) or polypeptide in accordancewith the method in the present invention can be continuous orintermittent, depending, for example, upon the recipient's physiologicalcondition, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an antibody may be essentially continuous over apreselected period of time or may be in a series of spaced dose, e.g.,either before, during, or after developing pain, before, during, beforeand after, during and after, or before, during, and after developingpain. Administration can be before, during and/or after wound, incision,trauma, surgery, and any other event likely to give rise topost-surgical pain.

Other formulations include suitable delivery forms known in the artincluding, but not limited to, carriers such as liposomes. See, forexample, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomalpreparations include, but are not limited to, cytofectins, multilamellarvesicles and unilamellar vesicles.

In some embodiments, more than one antibody or polypeptide may bepresent. The antibodies can be monoclonal or polyclonal. Suchcompositions may contain at least one, at least two, at least three, atleast four, at least five different antibodies. A mixture of antibodies,as they are often denoted in the art, may be particularly useful intreating a broader range of population of individuals.

A polynucleotide encoding any of the antibodies or polypeptides of theinvention (such as antibody E3) may also be used for delivery andexpression of any of the antibodies or polypeptides of the invention(such as antibody E3) in a desired cell. It is apparent that anexpression vector can be used to direct expression of an E3 antibody orpolypeptide. The expression vector can be administered by any meansknown in the art, such as intraperitoneally, intravenously,intramuscularly, subcutaneously, intrathecally, intraventricularly,orally, enterally, parenterally, intranasally, dermally, sublingually,or by inhalation. For example, administration of expression vectorsincludes local or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. One skilled in the art is familiar with administrationof expression vectors to obtain expression of an exogenous protein invivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.

Targeted delivery of therapeutic compositions comprising apolynucleotide encoding any of the antibodies or polypeptides of theinvention (such as antibody E3) can also be used. Receptor-mediated DNAdelivery techniques are described in, for example, Findeis et al.,Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics:Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.)(1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol.Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. (USA) (1990)87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides of the present inventioncan be delivered using gene delivery vehicles. The gene delivery vehiclecan be of viral or non-viral origin (see generally, Jolly, Cancer GeneTherapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly,Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994)6:148). Expression of such coding sequences can be induced usingendogenous mammalian or heterologous promoters. Expression of the codingsequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740; 4,777,127; GB Patent No. 2,200,651; and EP PatentNo. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors,Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCCVR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCCVR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associatedvirus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655).Administration of DNA linked to killed adenovirus as described inCuriel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent NO. 0 524 968.Additional approaches are described in Philip, Mol. Cell Biol. (1994)14:2411 and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

With respect to all methods described herein, reference to anti-NGFantagonist antibodies also include compositions comprising one or moreof these agents. These compositions may further comprise suitableexcipients, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art. The present invention can beused alone or in combination with other conventional methods oftreatment.

Methods of Using Anti-NGF Antagonist Antibody for Treating or PreventingRheumatoid Arthritis Pain

In some aspects, the invention provides methods for treating and/orpreventing rheumatoid arthritis pain in individuals including mammals,both human and non-human. Accordingly, in one aspect, the inventionprovides methods of treating rheumatoid arthritis pain in an individualcomprising administering an effective amount of an anti-NGF antagonistantibody. Anti-NGF antagonist antibodies are known in the art anddescribed herein.

In another aspect, the invention provides methods for reducing incidenceof, ameliorating, suppressing, palliating, and/or delaying the onset,the development or the progression of rheumatoid arthritis pain in anindividual. Thus, in some embodiments, the anti-NGF antagonist antibodyis administered prior to development of pain or a pain episode in anindividual having rheumatoid arthritis.

In another aspect, the invention provides methods for treatinginflammatory cachexia (weight loss) associated with rheumatoid arthritisin an individual comprising administering an effective amount of ananti-NGF antagonist antibody (Roubenoff et al., Arthritis Rheum. 40(3):534-9 (1997); Roubenoff et al., J. Clin. Invest. 93(6):2379-86 (1994)).

Diagnosis or assessment of rheumatoid arthritis pain is well-establishedin the art. Assessment may be performed based on measures known in theart, such as patient characterization of pain using various pain scales.See, e.g., Katz et al, Surg Clin North Am. (1999) 79 (2):231-52;Caraceni et al. J Pain Symptom Manage (2002) 23(3):239-55. There arealso commonly used scales to measure disease state such as the AmericanCollege of Rheumatology (ACR) (Felson, et al., Arthritis and Rheumatism(1993) 36(6):729-740), the Health Assessment Questionnaire (HAQ) (Fries,et al., (1982) J. Rheumatol. 9: 789-793), the Paulus Scale (Paulus, etal., Arthritis and Rheumatism (1990) 33: 477-484), and the ArthritisImpact Measure Scale (AIMS) (Meenam, et al., Arthritis and Rheumatology(1982) 25: 1048-1053). Anti-NGF antagonist antibody may be administeredto an individual via any suitable route. Examples of differentadministration route are described herein.

Pain relief may be characterized by time course of relief. Accordingly,in some embodiments, pain relief is observed within about 24 hours afteradministration of anti-NGF antagonist antibody. In other embodiments,pain relief is observed within about 36, 48, 60, 72 hours or 4 daysafter administration of anti-NGF antagonist antibody. In still otherembodiments, pain relief is observed before observing an indication ofimprovement of the inflammatory condition associated with rheumatoidarthritis. In some embodiments, frequency and/or intensity of pain isdiminished, and/or quality of life of those suffering the disease isincreased.

Making and using NGF antagonists (including anti-NGF antibodies) forthese methods are described in sections below (“NGF antagonists”,“Anti-NGF antagonist antibody”; “Other NGF antagonists”; “Identificationof NGF antagonists (such as anti-NGF antagonist antibodies)”;“Compositions for use in the methods of the invention”; “Administrationof an NGF antagonist (such as an anti-NGF antagonist antibody)”).

Methods of Using Anti-NGF Antagonist Antibody for Treating or PreventingOsteoarthritis Pain

In some aspects, the invention provides methods for treating and/orpreventing osteoarthritis pain in individuals including mammals, bothhuman and non-human. Accordingly, in one aspect, the invention providesmethods of treating osteoarthritis pain in an individual comprisingadministering an effective amount of an NGF antagonist (such as ananti-NGF antagonist antibody). NGF antagonists, including anti-NGFantagonist antibodies, are known in the art and described herein.

In another aspect, the invention provides methods for reducing incidenceof, ameliorating, suppressing, palliating, and/or delaying the onset,the development or the progression of osteoarthritis pain in anindividual comprising administering an effective amount of an NGFantagonist (such as an anti-NGF antagonist antibody). Thus, in someembodiments, the NGF antagonist (such as anti-NGF antagonist antibody)is administered prior to development of pain or a pain episode in anindividual having osteoarthritis.

Diagnosis or assessment of osteoarthritis pain is well-established inthe art. Assessment may be performed based on measures known in the art,such as patient characterization of pain using various pain scales. See,e.g., Katz et al, Surg Clin North Am. (1999) 79 (2):231-52; Caraceni etal. J Pain Symptom Manage (2002) 23(3):239-55. For example, WOMACAmbulation Pain Scale (including pain, stiffness, and physical function)and 100 mm Visual Analogue Scale (VAS) may be employed to assess painand evaluate response to the treatment.

NGF antagonists (such as an anti-NGF antagonist antibody) may beadministered to an individual via any suitable route. Examples ofdifferent administration route are described herein.

In some embodiments, the NGF antagonist (such as the anti-NGF antagonistantibody) is administered once every week, once every two weeks, onceevery three weeks, once every four weeks, once every five weeks, onceevery six weeks, once every seven weeks, once every eight weeks, onceevery nine weeks, once every ten weeks, once every fifteen weeks, onceevery twenty weeks, once every twenty five weeks, or once every twentysix weeks. In some embodiments, the NGF antagonist (such as the anti-NGFantagonist antibody) is administered once every month, once every twomonths, once every three months, once every four months, once every fivemonths, or once every six months.

Pain relief may be characterized by time course of relief. Accordingly,in some embodiments, pain relief is observed within about 24 hours afteradministration of an NGF antagonist (such as an anti-NGF antagonistantibody). In other embodiments, pain relief is observed within about36, 48, 60, 72 hours or 4 days after administration of the NGFantagonist (such as the anti-NGF antagonist antibody). In someembodiments, frequency and/or intensity of pain is diminished, and/orquality of life of those suffering the disease is increased. In someembodiments, pain relief for osteoarthritis is provided for duration ofat least about 7 days, at least about 14 days, at least about 21 days,at least about 28 days, at least about 35 days, at least about 42 days,at least about 49 days, at least about 56 days, at least about 63 days,at least about 70 days, at least about 77 days, at least about 84 days,at least about 180 days, or longer after a single dose of the NGFantagonist (such as the anti-NGF antagonist antibody).

Making and using NGF antagonists (including anti-NGF antibodies) forthese methods are described in sections below (“NGF antagonists”,“Anti-NGF antagonist antibody”; “Other NGF antagonists”; “Identificationof NGF antagonists (such as anti-NGF antagonist antibodies)”;“Compositions for use in the methods of the invention”; “Administrationof an NGF antagonist (such as an anti-NGF antagonist antibody)”).

NGF Antagonists

The methods of the invention (pertaining to rheumatoid arthritis painand osteoarthritis pain) use an NGF antagonist, which refers to anymolecule that blocks, suppresses or reduces (including significantly)NGF biological activity, including downstream pathways mediated by NGFsignaling, such as receptor binding and/or elicitation of a cellularresponse to NGF. The term “antagonist” implies no specific mechanism ofbiological action whatsoever, and is deemed to expressly include andencompass all possible pharmacological, physiological, and biochemicalinteractions with NGF and its consequences which can be achieved by avariety of different, and chemically divergent, compositions. ExemplaryNGF antagonists include, but are not limited to, an anti-NGF antibody,an anti-sense molecule directed to NGF (including an anti-sense moleculedirected to a nucleic acid encoding NGF), an anti-sense moleculedirected to an NGF receptor (such as TrkA receptor and/or p75 receptor)(including an anti-sense molecule directed to a nucleic acid encodingTrkA and/or p75), an NGF inhibitory compound, an NGF structural analog,a dominant-negative mutation of a TrkA receptor that binds an NGF, aTrkA immunoadhesin, an anti-TrkA antibody, a dominant-negative mutationof a p75 receptor that binds an NGF, an anti-p75 antibody, and a kinaseinhibitor. For purpose of the present invention, it will be explicitlyunderstood that the term “antagonist” encompasses all the previouslyidentified terms, titles, and functional states and characteristicswhereby the NGF itself, an NGF biological activity (including but notlimited to its ability to mediate any aspect of pain), or theconsequences of the biological activity, are substantially nullified,decreased, or neutralized in any meaningful degree. In some embodiments,an NGF antagonist (e.g., an antibody) binds (physically interact with)NGF, binds to an NGF receptor (such as TrkA receptor and/or p75receptor), and/or reduces (impedes and/or blocks) downstream NGFreceptor signaling. Accordingly, in some embodiments, an NGF antagonistbinds (physically interacts with) NGF. In some embodiments, the NGFantagonist is a polypeptide which binds to NGF. In some embodiments, theNGF antagonist is a peptide or a modified peptide (such as NGF bindingpeptide fused to a Fc domain) described in PCT WO 2004/026329. In otherembodiment, an NGF antagonist binds to an NGF receptor (such as trkAreceptor or p75). In other embodiments, an NGF antagonist reduces(impedes and/or blocks) downstream NGF receptor signaling (e.g.,inhibitors of kinase signaling). In other embodiments, an NGF antagonistinhibits (reduces) NGF synthesis and/or release. In another embodiment,the NGF antagonist is an NGF antagonist that is not a TrkA immunoadhesin(i.e., is other than a TrkA immunoadhesin). In another embodiment, theNGF antagonist is other than an anti-NGF antibody. In other embodiment,the NGF antagonist is other than a TrkA immunoadhesin and other than ananti-NGF antibody. In some embodiment, the NGF antagonist binds NGF(such as hNGF) and does not significantly bind to related neurotrophins,such as NT-3, NT4/5, and/or BDNF. In some embodiments, the NGFantagonist is not associated with an adverse immune response. In otherembodiments, the NGF antagonist is an anti-NGF antibody. In still otherembodiments, the anti-NGF antibody is humanized (such as antibody E3described herein). In some embodiments, the anti-NGF antibody isantibody E3 (as described herein). In other embodiments, the anti-NGFantibody comprises one or more CDR(s) of antibody E3 (such as one, two,three, four, five, or, in some embodiments, all six CDRs from E3). Inother embodiments, the antibody is human. In still other embodiments,the anti-NGF antibody comprises the amino acid sequence of the heavychain variable region shown in FIG. 1A (SEQ ID NO:1) and the amino acidsequence of the light chain variable region shown in FIG. 1B (SEQ IDNO:2). In still other embodiments, the antibody comprises a modifiedconstant region, such as a constant region that is immunologicallyinert, e.g., does not trigger complement mediated lysis, or does notstimulate antibody-dependent cell mediated cytotoxicity (ADCC). In otherembodiments, the constant region is modified as described in Eur. J.Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/orUK Patent Application No. 9809951.8.

Anti-NGF Antagonist Antibody

The methods of the invention (pertaining to rheumatoid arthritis painand osteoarthritis pain) use an anti-NGF antagonist antibody, whichrefers to any antibody molecule that blocks, suppresses or reduces(including significantly) NGF biological activity, including downstreampathways mediated by NGF signaling, such as receptor binding and/orelicitation of a cellular response to NGF.

An anti-NGF antagonist antibody should exhibit any one or more of thefollowing characteristics: (a) bind to NGF and inhibit NGF biologicalactivity or downstream pathways mediated by NGF signaling function; (b)prevent, ameliorate, or treat any aspect of rheumatoid arthritis pain orosteoarthritis pain; (c) block or decrease NGF receptor activation(including TrkA receptor dimerization and/or autophosphorylation); (d)increase clearance of NGF; (e) inhibit (reduce) NGF synthesis,production or release. Anti-NGF antagonist antibodies are known in theart, see, e.g., PCT Publication Nos. WO 01/78698, WO 01/64247, U.S. Pat.Nos. 5,844,092, 5,877,016, and 6,153,189; Hongo et al., Hybridoma,19:215-227 (2000); Cell. Molec. Biol. 13:559-568 (1993); GenBankAccession Nos. U39608, U39609, L17078, or L17077. Anti-NGF antagonistantibodies and polypeptides are also described in PCT WO 2005/019266.

For purposes of this invention, the antibody reacts with NGF in a mannerthat inhibits NGF and/or downstream pathways mediated by the NGFsignaling function. In some embodiments, the anti-NGF antagonistantibody recognizes human NGF. In yet other embodiments, the anti-NGFantagonist antibody specifically binds human NGF. In some embodiment,the anti-NGF antagonist antibody does not significantly bind to relatedneurotrophins, such as NT-3, NT4/5, and/or BDNF. In still otherembodiments, the anti-NGF antibody is capable of binding NGF andeffectively inhibiting the binding of NGF to its TrkA and/or p75receptor in vivo and/or effectively inhibiting NGF from activating itsTrkA and/or p75 receptor. In still other embodiments, the anti-NGFantagonist antibody is a monoclonal antibody. In still otherembodiments, the anti-NGF antibody is humanized (such as antibody E3described herein). In some embodiments, the anti-NGF antibody is human.See, e.g., WO 2005/019266. In one embodiment, the antibody is a humanantibody which recognizes one or more epitopes on human NGF. In anotherembodiment, the antibody is a mouse or rat antibody which recognizes oneor more epitopes on human NGF. In another embodiment, the antibodyrecognizes one or more epitopes on an NGF selected from the groupconsisting of: primate, canine, feline, equine, and bovine. In stillfurther embodiments, the anti-NGF antagonist antibody binds essentiallythe same NGF epitope 6 as an antibody selected from any one or more ofthe following: MAb 911, MAb 912 and MAb 938 (See Hongo, et al.,Hybridoma 19:215-227 (2000)). In other embodiments, the antibody bindsthe same epitope as Mab 911. In another embodiment, the antibodycomprises a constant region that is immunologically inert (e.g., doesnot trigger complement mediated lysis or antibody dependent cellmediated cytotoxicity (ADCC)). ADCC activity can be assessed usingmethods disclosed in U.S. Pat. No. 5,500,362. In some embodiments, theconstant region is modified as described in Eur. J. Immunol. (1999)29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK PatentApplication No. 9809951.8.

In some embodiments, the anti-NGF antagonist antibody is a humanizedmouse anti-NGF monoclonal antibody termed antibody “E3”, any of the E3related antibodies described herein, or any fragments thereof, which areNGF antagonists.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)₂, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion, humanized antibodies, and anyother modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity,including glycosylation variants of antibodies, amino acid sequencevariants of antibodies, and covalently modified antibodies. Theantibodies may be murine, rat, human, or any other origin (includingchimeric or humanized antibodies).

The binding affinity of an anti-NGF antagonist antibody to NGF (such ashNGF) can be about 0.10 to about 0.80 nM, about 0.15 to about 0.75 nMand about 0.18 to about 0.72 nM. In one embodiment, the binding affinityis between about 2 pM and 22 pM. In one embodiment, the binding affinityis between about 23 pM and about 100 pM. In some embodiment, the bindingaffinity is about 10 nM. In other embodiments, the binding affinity isless than about 10 nM. In other embodiments, the binding affinity isabout 0.1 nM or about 0.07 nM. In other embodiments, the bindingaffinity is less than about 0.1 nM or less than about 0.07 nM. In otherembodiments, the binding affinity is any of about 100 nM, about 50 nM,about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM toany of about 2 pM, about 5 pM, about 10 pM, about 15 pM, about 20 pM, orabout 40 pM. In some embodiments, the binding affinity is any of about100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100pM, or about 50 pM, or less than about 50 pM. In some embodiments, thebinding affinity is less than any of about 100 nM, about 50 nM, about 10nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM. In stillother embodiments, the binding affinity is about 2 pM, about 5 pM, about10 pM, about 15 pM, about 20 pM, about 40 pM, or greater than about 40pM.

One way of determining binding affinity of antibodies to NGF is bymeasuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of an anti-NGF Fab fragment of an antibody can be determined bysurface plasmon resonance (BIAcore3000™ surface plasmon resonance (SPR)system, BIAcore, INC, Piscaway N.J.). CM5 chips can be activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF (or any other NGF) can be diluted into 10 mM sodium acetate pH4.0 and injected over the activated chip at a concentration of 0.005mg/mL. Using variable flow time across the individual chip channels, tworanges of antigen density can be achieved: 100-200 response units (RU)for detailed kinetic studies and 500-600 RU for screening assays. Thechip can be blocked with ethanolamine. Regeneration studies have shownthat a mixture of Pierce elution buffer (Product No. 21004, PierceBiotechnology, Rockford Ill.) and 4 M NaCl (2:1) effectively removes thebound Fab while keeping the activity of hNGF on the chip for over 200injections. HBS-EP buffer (0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA,0.005% Surfactant P29) is used as running buffer for the BIAcore assays.Serial dilutions (0.1-10× estimated K_(D)) of purified Fab samples areinjected for 1 min at 100 QXIL/min and dissociation times of up to 2 hare allowed. The concentrations of the Fab proteins are determined byELISA and/or SDS-PAGE electrophoresis using a Fab of known concentration(as determined by amino acid analysis) as a standard. Kineticassociation rates (kon) and dissociation rates (koff) are obtainedsimultaneously by fitting the data to a 1:1 Langmuir binding model(Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values are calculated as k_(off)/k_(on).This protocol is suitable for use in determining binding affinity of anantibody to any NGF, including human NGF, NGF of another vertebrate (insome embodiments, mammalian) (such as mouse NGF, rat NGF, primate NGF),as well as for use with other neurotrophins, such as the relatedneurotrophins NT3, NT4/5, and/or BDNF.

In some embodiments, the antibody binds human NGF, and does notsignificantly bind an NGF from another vertebrate species (in someembodiment, mammalian). In some embodiments, the antibody binds humanNGF as well as one or more NGF from another vertebrate species (in someembodiments, mammalian). In still other embodiments, the antibody bindsNGF and does not significantly cross-react with other neurotrophins(such as the related neurotrophins, NT3, NT4/5, and/or BDNF). In someembodiments, the antibody binds NGF as well as at least one otherneurotrophin. In some embodiments, the antibody binds to a mammalianspecies of NGF, such as horse or dog, but does not significantly bind toNGF from anther mammalian species.

The epitope(s) can be continuous or discontinuous. In one embodiment,the antibody binds essentially the same hNGF epitopes as an antibodyselected from the group consisting of MAb 911, MAb 912, and MAb 938 asdescribed in Hongo et al., Hybridoma, 19:215-227 (2000). In anotherembodiment, the antibody binds essentially the same hNGF epitope as MAb911. In still another embodiment, the antibody binds essentially thesame epitope as MAb 909. Hongo et al., supra. For example, the epitopemay comprise one or more of: residues K32, K34 and E35 within variableregion 1 (amino acids 23-35) of hNGF; residues F79 and T81 withinvariable region 4 (amino acids 81-88) of hNGF; residues H84 and K88within variable region 4; residue R103 between variable region 5 (aminoacids 94-98) of hNGF and the C-terminus (amino acids 111-118) of hNGF;residue E11 within pre-variable region 1 (amino acids 10-23) of hNGF;Y52 between variable region 2 (amino acids 40-49) of hNGF and variableregion 3 (amino acids 59-66) of hNGF; residues L112 and S113 within theC-terminus of hNGF; residues R59 and R69 within variable region 3 ofhNGF; or residues V18, V20, and G23 within pre-variable region 1 ofhNGF. In addition, an epitope can comprise one or more of the variableregion 1, variable region 3, variable region 4, variable region 5, theN-terminus region, and/or the C-terminus of hNGF. In still anotherembodiment, the antibody significantly reduces the solvent accessibilityof residue R103 of hNGF. It is understood that although the epitopesdescribed above relate to human NGF, one of ordinary skill can align thestructures of human NGF with the NGF of other species and identifylikely counterparts to these epitopes.

In one aspect, antibodies (e.g., human, humanized, mouse, chimeric) thatcan inhibit NGF may be made by using immunogens that express full lengthor partial sequence of NGF. In another aspect, an immunogen comprising acell that overexpresses NGF may be used. Another example of an immunogenthat can be used is NGF protein that contains full-length NGF or aportion of the NGF protein.

The anti-NGF antagonist antibodies may be made by any method known inthe art. The route and schedule of immunization of the host animal aregenerally in keeping with established and conventional techniques forantibody stimulation and production, as further described herein.General techniques for production of human and mouse antibodies areknown in the art and are described herein.

It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human, hybridoma celllines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the anti-NGF monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for NGF, or a portion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a human NGF, or a fragmentcontaining the target amino acid sequence conjugated to a protein thatis immunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for examplemaleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glytaradehyde, succinic anhydride, SOCl2, or R1N═C═NR, where R and R1are different alkyl groups, can yield a population of antibodies (e.g.,monoclonal antibodies).

If desired, the anti-NGF antagonist antibody (monoclonal or polyclonal)of interest may be sequenced and the polynucleotide sequence may then becloned into a vector for expression or propagation. The sequenceencoding the antibody of interest may be maintained in vector in a hostcell and the host cell can then be expanded and frozen for future use.In an alternative, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. For example, the constant regionmay be engineered to more resemble human constant regions to avoidimmune response if the antibody is used in clinical trials andtreatments in humans. It may be desirable to genetically manipulate theantibody sequence to obtain greater affinity to NGF and greater efficacyin inhibiting NGF. It will be apparent to one of skill in the art thatone or more polynucleotide changes can be made to the anti-NGFantagonist antibody and still maintain its binding ability to NGF.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains (2) designing thehumanized antibody, i.e., deciding which antibody framework region touse during the humanizing process (3) the actual humanizingmethodologies/techniques and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; and 6,180,370.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated complementarity determining regions (CDRs) fused tohuman constant domains. See, for example, Winter et al. Nature349:293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA86:4220-4224 (1989), Shaw et al. J Immunol. 138:4534-4538 (1987), andBrown et al. Cancer Res. 47:3577-3583 (1987). Other references describerodent CDRs grafted into a human supporting framework region (FR) priorto fusion with an appropriate human antibody constant domain. See, forexample, Riechmann et al. Nature 332:323-327 (1988), Verhoeyen et al.Science 239:1534-1536 (1988), and Jones et al. Nature 321:522-525(1986). Another reference describes rodent CDRs supported byrecombinantly veneered rodent framework regions. See, for example,European Patent Publication No. 0519596. These “humanized” molecules aredesigned to minimize unwanted immunological response toward rodentanti-human antibody molecules which limits the duration andeffectiveness of therapeutic applications of those moieties in humanrecipients. For example, the antibody constant region can be engineeredsuch that it is immunologically inert (e.g., does not trigger complementlysis). See, e.g. PCT Publication No. PCT/GB99/01441; UK PatentApplication No. 9809951.8. Other methods of humanizing antibodies thatmay also be utilized are disclosed by Daugherty et al., Nucl. Acids Res.19:2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297;5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT PublicationNo. WO 01/27160.

In yet another alternative, fully human antibodies may be obtained byusing commercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. In another alternative, antibodiesmay be made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, thephage display technology (McCafferty et al., Nature 348:552-553 (1990))can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J.,Current Opinion in Structural Biology 3:564-571 (1993). Several sourcesof V-gene segments can be used for phage display. Clackson et al.,Nature 352:624-628 (1991) isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genes derivedfrom the spleens of immunized mice. A repertoire of V genes fromunimmunized human donors can be constructed and antibodies to a diversearray of antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Mark et al., J. Mol. Biol.222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). In anatural immune response, antibody genes accumulate mutations at a highrate (somatic hypermutation). Some of the changes introduced will conferhigher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling.” Marks, et al.,Bio/Technol. 10:779-783 (1992)). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thepM-nM range. A strategy for making very large phage antibody repertoires(also known as “the mother-of-all libraries”) has been described byWaterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993). Gene shufflingcan also be used to derive human antibodies from rodent antibodies,where the human antibody has similar affinities and specificities to thestarting rodent antibody. According to this method, which is alsoreferred to as “epitope imprinting”, the heavy or light chain V domaingene of rodent antibodies obtained by phage display technique isreplaced with a repertoire of human V domain genes, creatingrodent-human chimeras. Selection on antigen results in isolation ofhuman variable regions capable of restoring a functional antigen-bindingsite, i.e., the epitope governs (imprints) the choice of partner. Whenthe process is repeated in order to replace the remaining rodent Vdomain, a human antibody is obtained (see PCT Publication No. WO93/06213, published Apr. 1, 1993). Unlike traditional humanization ofrodent antibodies by CDR grafting, this technique provides completelyhuman antibodies, which have no framework or CDR residues of rodentorigin.

It is apparent that although the above discussion pertains to humanizedantibodies, the general principles discussed are applicable tocustomizing antibodies for use, for example, in dogs, cats, primate,equines and bovines. It is further apparent that one or more aspects ofhumanizing an antibody described herein may be combined, e.g., CDRgrafting, framework mutation and CDR mutation.

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756 (2001); Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65 (1995); and Pollock, et al., J Immunol Methods231:147(1999). Methods for making derivatives of antibodies, e.g.,humanized, single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for NGF.

The antibodies can be bound to many different carriers. Carriers can beactive and/or inert. Examples of well-known carriers includepolypropylene, polystyrene, polyethylene, dextran, nylon, amylases,glass, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation. In some embodiments, thecarrier comprises a moiety that targets the myocardium.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors (such as expression vectors disclosed in PCTPublication No. WO 87/04462), which are then transfected into host cellssuch as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. See, e.g., PCT Publication No. WO 87/04462. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci.81:6851 (1984), or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of an anti-NGF monoclonalantibody herein.

Anti-NGF antagonist antibodies may be characterized using methods wellknown in the art. For example, one method is to identify the epitope towhich it binds, or “epitope mapping.” There are many methods known inthe art for mapping and characterizing the location of epitopes onproteins, including solving the crystal structure of an antibody-antigencomplex, competition assays, gene fragment expression assays, andsynthetic peptide-based assays, as described, for example, in Chapter 11of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In anadditional example, epitope mapping can be used to determine thesequence to which an anti-NGF antagonist antibody binds. Epitope mappingis commercially available from various sources, for example, PepscanSystems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitopecan be a linear epitope, i.e., contained in a single stretch of aminoacids, or a conformational epitope formed by a three-dimensionalinteraction of amino acids that may not necessarily be contained in asingle stretch. Peptides of varying lengths (e.g., at least 4-6 aminoacids long) can be isolated or synthesized (e.g., recombinantly) andused for binding assays with an anti-NGF antagonist antibody. In anotherexample, the epitope to which the anti-NGF antagonist antibody binds canbe determined in a systematic screening by using overlapping peptidesderived from the NGF sequence and determining binding by the anti-NGFantagonist antibody. According to the gene fragment expression assays,the open reading frame encoding NGF is fragmented either randomly or byspecific genetic constructions and the reactivity of the expressedfragments of NGF with the antibody to be tested is determined. The genefragments may, for example, be produced by PCR and then transcribed andtranslated into protein in vitro, in the presence of radioactive aminoacids. The binding of the antibody to the radioactively labeled NGFfragments is then determined by immunoprecipitation and gelelectrophoresis. Certain epitopes can also be identified by using largelibraries of random peptide sequences displayed on the surface of phageparticles (phage libraries). Alternatively, a defined library ofoverlapping peptide fragments can be tested for binding to the testantibody in simple binding assays. In an additional example, mutagenesisof an antigen binding domain, domain swapping experiments and alaninescanning mutagenesis can be performed to identify residues required,sufficient, and/or necessary for epitope binding. For example, domainswapping experiments can be performed using a mutant NGF in whichvarious fragments of the NGF polypeptide have been replaced (swapped)with sequences from a closely related, but antigenically distinctprotein (such as another member of the neurotrophin protein family). Byassessing binding of the antibody to the mutant NGF, the importance ofthe particular NGF fragment to antibody binding can be assessed.

Yet another method which can be used to characterize an anti-NGFantagonist antibody is to use competition assays with other antibodiesknown to bind to the same antigen, i.e., various fragments on NGF, todetermine if the anti-NGF antagonist antibody binds to the same epitopeas other antibodies. Competition assays are well known to those of skillin the art. Example of antibodies that can be used in the competitionassays for the present invention include MAb 911, 912, 938, as describedin Hongo, et al., Hybridoma 19:215-227 (2000).

An expression vector can be used to direct expression of an anti-NGFantagonist antibody. One skilled in the art is familiar withadministration of expression vectors to obtain expression of anexogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471. Administration of expression vectors includeslocal or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. In another embodiment, the expression vector isadministered directly to the sympathetic trunk or ganglion, or into acoronary artery, atrium, ventricle, or pericardium.

Targeted delivery of therapeutic compositions containing an expressionvector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer (J. A.Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al.,J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA(1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51;Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy(1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, andin Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

Other NGF Antagonists

NGF antagonists other than anti-NGF antibodies may be used (pertainingto rheumatoid arthritis pain and osteoarthritis pain). In someembodiments of the invention, the NGF antagonist comprises at least oneantisense molecule capable of blocking or decreasing the expression of afunctional NGF. Nucleotide sequences of the NGF are known and arereadily available from publicly available databases. See, e.g., Borsaniet al., Nuc. Acids Res. 1990, 18, 4020; Accession Number NM 002506;Ullrich et al., Nature 303:821-825 (1983). It is routine to prepareantisense oligonucleotide molecules that will specifically bind NGF mRNAwithout cross-reacting with other polynucleotides. Exemplary sites oftargeting include, but are not limited to, the initiation codon, the 5′regulatory regions, the coding sequence and the 3′ untranslated region.In some embodiments, the oligonucleotides are about 10 to 100nucleotides in length, about 15 to 50 nucleotides in length, about 18 to25 nucleotides in length, or more. The oligonucleotides can comprisebackbone modifications such as, for example, phosphorothioate linkages,and 2′-0 sugar modifications well know in the art. Exemplary antisensemolecules include the NGF antisense molecules described in U.S.Publication No. 20010046959; see also http://www.rna-tec.com/repair.htm.

In other embodiments, the NGF antagonist comprises at least oneantisense molecule capable of blocking or decreasing the expression of afunctional NGF receptor (such as TrkA and/or p75). Woolf et al., J.Neurosci. (2001) 21(3):1047-55; Taglialetela et al, J Neurochem (1996)66(5): 1826-35. Nucleotide sequences of TrkA and p75 are known and arereadily available from publicly available databases.

Alternatively, NGF expression and/or release and/or NGF receptorexpression can be decreased using gene knockdown, morpholinooligonucleotides, RNAi, or ribozymes, methods that are well-known in theart. See http://www.macalester.edu/˜montgomery/RNAi.html;http://pub32.ezboard.com/fmorpholinosfrm19.showMessage?topicID=6.topic;http://www.highveld.com/ribozyme.html.

In other embodiments, the NGF antagonist comprises at least one NGFinhibitory compound. As used herein, “NGF inhibitory compound” refers toa compound other than an anti-NGF antibody that directly or indirectlyreduces, inhibits, neutralizes, or abolishes NGF biological activity. AnNGF inhibitory compound should exhibit any one or more of the followingcharacteristics: (a) bind to NGF and inhibit NGF biological activityand/or downstream pathways mediated by NGF signaling function; (b)prevent, ameliorate, or treat any aspect of pain (such as osteoarthritispain); (c) block or decrease NGF receptor activation (including TrkAreceptor dimerization and/or autophosphorylation); (d) increaseclearance of NGF; (e) inhibit (reduce) NGF synthesis, production orrelease. Exemplary NGF inhibitory compounds include the small moleculeNGF inhibitors described in U.S. Publication No. 20010046959; thecompounds that inhibit NGF's binding to p75, as described in PCTPublication No. WO 00/69829, and PD90780[7-(benzolylamino)-4,9-dihydro-4-methyl-9-oxo-pyrazolo[5,1-b]quinazoline-2-carboxylicacid] as described by Colquhoun et al., J. Pharmacol. Exp. Ther.310(2):505-11 (2004); the compounds that inhibit NGF's binding to TrkAand/or p75, as described in PCT Publication No. WO 98/17278. Additionalexamples of NGF inhibitory compounds include the compounds described inPCT Publication Nos. WO 02/17914 and WO 02/20479, and in U.S. Pat. Nos.5,342,942; 6,127,401; and 6,359,130. Further exemplary NGF inhibitorycompounds are compounds that are competitive inhibitors of NGF. See U.S.Pat. No. 6,291,247. Furthermore, one skilled in the art can prepareother small molecules NGF inhibitory compounds.

In some embodiments, an NGF inhibitory compound binds NGF. Exemplarysites of targeting (binding) include, but are not limited to, theportion of the NGF that binds to the TrkA receptor and/or p75 receptor,and those portions of the NGF that are adjacent to the receptor-bindingregion and which are responsible, in part, for the correctthree-dimensional shape of the receptor-binding portion. In anotherembodiment, an NGF inhibitory compound binds an NGF receptor (such asTrkA and/or p75) and inhibits an NGF biological activity. Exemplarysites of targeting include those portions of TrkA and/or p75 that bindto NGF.

In embodiments comprising small molecules, a small molecule can have amolecular weight of about any of 100 to 20,000 daltons, 500 to 15,000daltons, or 1000 to 10,000 daltons. Libraries of small molecules arecommercially available. The small molecules can be administered usingany means known in the art, including inhalation, intraperitoneally,intravenously, intramuscularly, subcutaneously, intrathecally,intraventricularly, orally, enterally, parenterally, intranasally, ordermally. In general, when the NGF-antagonist according to the inventionis a small molecule, it will be administered at the rate of 0.1 to 300mg/kg of the weight of the patient divided into one to three or moredoses. For an adult patient of normal weight, doses ranging from 1 mg to5 g per dose can be administered.

In other embodiments, the NGF antagonist comprises at least one NGFstructural analog. “NGF structural analogs” in the present inventionrefer to compounds that have a similar 3-dimensional structure as partof that of NGF and which bind to an NGF receptor under physiologicalconditions in vitro or in vivo, wherein the binding at least partiallyinhibits an NGF biological activity. In one embodiment, the NGFstructural analog binds to a TrkA and/or a p75 receptor. Exemplary NGFstructural analogs include, but are not limited to, the bicyclicpeptides described in PCT Publication No. WO 97/15593; the bicyclicpeptides described in U.S. Pat. No. 6,291,247; the cyclic compoundsdescribed in U.S. Pat. No. 6,017,878; and NGF-derived peptides describedin PCT Publication No. WO 89/09225. Suitable NGF structural analogs canalso be designed and synthesized through molecular modeling ofNGF-receptor binding, for example by the method described in PCTPublication No. WO 98/06048. The NGF structural analogs can be monomersor dimers/oligomers in any desired combination of the same or differentstructures to obtain improved affinities and biological effects.

In other embodiments, the invention provides an NGF antagonistcomprising at least one dominant-negative mutant of the TrkA receptorand/or p75 receptor. One skilled in the art can preparedominant-negative mutants of, e.g., the TrkA receptor such that thereceptor will bind the NGF and, thus, act as a “sink” to capture NGFs.The dominant-negative mutants, however, will not have the normalbioactivity of the TrkA receptor upon binding to NGF. Exemplarydominant-negative mutants include, but are not limited to, the mutantsdescribed in the following references: Li et al., Proc. Natl. Acad. Sci.USA 1998, 95, 10884; Eide et al., J. Neurosci. 1996, 16, 3123; Liu etal., J. Neurosci 1997, 17, 8749; Klein et al., Cell 1990, 61, 647;Valenzuela et al., Neuron 1993, 10, 963; Tsoulfas et al., Neuron 1993,10, 975; and Lamballe et al., EMBO J. 1993, 12, 3083, each of which isincorporated herein by reference in its entirety. The dominant negativemutants can be administered in protein form or in the form of anexpression vector such that the dominant negative mutant, e.g., mutantTrkA receptor, is expressed in vivo. The protein or expression vectorcan be administered using any means known in the art, such asintraperitoneally, intravenously, intramuscularly, subcutaneously,intrathecally, intraventricularly, orally, enterally, parenterally,intranasally, dermally, or by inhalation. For example, administration ofexpression vectors includes local or systemic administration, includinginjection, oral administration, particle gun or catheterizedadministration, and topical administration. One skilled in the art isfamiliar with administration of expression vectors to obtain expressionof an exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338. Therapeutic compositions containing a polynucleotide areadministered in a range of about 100 ng to about 200 mg of DNA for localadministration in a gene therapy protocol. In some embodiments,concentration ranges of about 500 ng to about 50 mg, about 1 μg to about2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNAor more can also be used during a gene therapy protocol. The therapeuticpolynucleotides and polypeptides of the present invention can bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

It is also apparent that an expression vector can be used to directexpression of any of the protein-based NGF antagonists described herein(e.g., anti-NGF antibody, TrkA immunoadhesin, etc.). For example, otherTrkA receptor fragments that are capable of blocking (from partial tocomplete blocking) NGF and/or an NGF biological activity are known inthe art.

In another embodiment, the NGF antagonist comprises at least one TrkAimmunoadhesin. TrkA immunoadhesins as used herein refer to solublechimeric molecules comprising the extracellular domain of a TrkAreceptor and an immunoglobulin sequence, which retains the bindingspecificity of the TrkA receptor (substantially retains the bindingspecificity of the trkA receptor) and is capable of binding to NGF.

TrkA immunoadhesins are known in the art, and have been found to blockthe binding of NGF to the TrkA receptor. See, e.g., U.S. Pat. No.6,153,189. Brennan et al. report administration of TrkA immunoadhesin ina rat model of post-surgical pain. See Society for NeuroscienceAbstracts 24 (1-2) 880 (1998). In one embodiment, the TrkA immunoadhesincomprises a fusion of a TrkA receptor amino acid sequence (or a portionthereof) from TrkA extracellular domain capable of binding NGF (in someembodiments, an amino acid sequence that substantially retains thebinding specificity of the trkA receptor) and an immunoglobulinsequence. In some embodiments, the TrkA receptor is a human TrkAreceptor sequence, and the fusion is with an immunoglobulin constantdomain sequence. In other embodiments, the immunoglobulin constantdomain sequence is an immunoglobulin heavy chain constant domainsequence. In other embodiments, the association of two TrkAreceptor-immunoglobulin heavy chain fusions (e.g., via covalent linkageby disulfide bond(s)) results in a homodimeric immunoglobulin-likestructure. An immunoglobulin light chain can further be associated withone or both of the TrkA receptor-immunoglobulin chimeras in thedisulfide-bonded dimer to yield a homotrimeric or homotetramericstructure. Examples of suitable TrkA immunoadhesins include thosedescribed in U.S. Pat. No. 6,153,189.

In another embodiment, the NGF antagonist comprises at least oneanti-TrkA antibody capable of blocking, suppressing, altering, and/orreducing NGF physical interaction with the TrkA receptor and/ordownstream signaling, whereby an NGF biological activity is reducedand/or blocked. Anti-TrkA antibodies are known in the art. Exemplaryanti-TrkA antibodies include those described in PCT Publication Nos. WO97/21732, WO 00/73344, WO 02/15924, and U.S. Publication No.20010046959.

In another embodiment, the NGF antagonist comprises at least oneanti-p75 antibody capable of blocking, suppressing and/or reducing NGFphysical interaction with the p75 receptor and/or downstream signaling,whereby an NGF biological activity is reduced and/or blocked.

In another embodiment, the NGF antagonist comprises at least one kinaseinhibitor capable of inhibiting downstream kinase signaling associatedwith TrkA and/or p75 receptor activity. An exemplary kinase inhibitor isK252a or K252b, which is known in the art and described in Knusel etal., J. Neurochem. 59:715-722 (1992); Knusel et al., J. Neurochemistry57:955-962 (1991); Koizumi et al., J. Neuroscience 8:715-721 (1988);Hirata et al., Chemical Abstracts 111:728, XP00204135, see abstract and12th Collective Chemical Substance Index, p. 34237, c. 3 (5-7), 55-60,66-69), p. 34238, c.1 (41-44), c.2 (25-27, 32-33), p. 3423, c.3 (48-50,52-53); and U.S. Pat. No. 6,306,849.

It is expected that a number of other categories of NGF antagonists willbe identified if sought for by the clinician.

Identification of NGF Antagonists (Such as Anti-NGF AntagonistAntibodies)

NGF antagonists, including anti-NGF antagonist antibodies, can beidentified or characterized using methods known in the art, wherebyreduction, amelioration, or neutralization of an NGF biological activityis detected and/or measured. Methods described in PCT WO 04/065560 canbe used. Another method, for example, a kinase receptor activation(KIRA) assay described in U.S. Pat. Nos. 5,766,863 and 5,891,650, can beused to identify anti-NGF agents. This ELISA-type assay is suitable forqualitative or quantitative measurement of kinase activation bymeasuring the autophosphorylation of the kinase domain of a receptorprotein tyrosine kinase (hereinafter “rPTK”), e.g. TrkA receptor, aswell as for identification and characterization of potential antagonistsof a selected rPTK, e.g., TrkA. The first stage of the assay involvesphosphorylation of the kinase domain of a kinase receptor, for example,a TrkA receptor, wherein the receptor is present in the cell membrane ofan eukaryotic cell. The receptor may be an endogenous receptor ornucleic acid encoding the receptor, or a receptor construct, may betransformed into the cell. Typically, a first solid phase (e.g., a wellof a first assay plate) is coated with a substantially homogeneouspopulation of such cells (usually a mammalian cell line) so that thecells adhere to the solid phase. Often, the cells are adherent andthereby adhere naturally to the first solid phase. If a “receptorconstruct” is used, it usually comprises a fusion of a kinase receptorand a flag polypeptide. The flag polypeptide is recognized by thecapture agent, often a capture antibody, in the ELISA part of the assay.An analyte, such as a candidate NGF antagonist (including anti-NGFantagonist antibody) is then added together with NGF to the wells havingthe adherent cells, such that the tyrosine kinase receptor (e.g. TrkAreceptor) is exposed to (or contacted with) NGF and the analyte. Thisassay enables identification of antagonists (including antibodies) thatinhibit activation of TrkA by its ligand NGF. Following exposure to NGFand the analyte, the adhering cells are solubilized using a lysis buffer(which has a solubilizing detergent therein) and gentle agitation,thereby releasing cell lysate which can be subjected to the ELISA partof the assay directly, without the need for concentration orclarification of the cell lysate.

The cell lysate thus prepared is then ready to be subjected to the ELISAstage of the assay. As a first step in the ELISA stage, a second solidphase (usually a well of an ELISA microtiter plate) is coated with acapture agent (often a capture antibody) which binds specifically to thetyrosine kinase receptor, or, in the case of a receptor construct, tothe flag polypeptide. Coating of the second solid phase is carried outso that the capture agent adheres to the second solid phase. The captureagent is generally a monoclonal antibody, but, as is described in theexamples herein, polyclonal antibodies may also be used. The cell lysateobtained is then exposed to, or contacted with, the adhering captureagent so that the receptor or receptor construct adheres to (or iscaptured in) the second solid phase. A washing step is then carried out,so as to remove unbound cell lysate, leaving the captured receptor orreceptor construct. The adhering or captured receptor or receptorconstruct is then exposed to, or contacted with, an anti-phosphotyrosineantibody which identifies phosphorylated tyrosine residues in thetyrosine kinase receptor. In one embodiment, the anti-phosphotyrosineantibody is conjugated (directly or indirectly) to an enzyme whichcatalyses a color change of a non-radioactive color reagent.Accordingly, phosphorylation of the receptor can be measured by asubsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g.,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g., by a color change in the color reagent.

NGF antagonists (such as anti-NGF antagonist antibody) can also beidentified by incubating a candidate agent with NGF and monitoring anyone or more of the following characteristics: (a) binding to NGF andinhibiting NGF biological activity or downstream pathways mediated byNGF signaling function; (b) inhibiting, blocking or decreasing NGFreceptor activation (including TrkA dimerization and/orautophosphorylation); (c) increasing clearance of NGF; (d) treating orpreventing any aspect of rheumatoid arthritis pain or osteoarthritispain; (e) inhibiting (reducing) NGF synthesis, production or release. Insome embodiments, an NGF antagonist (e.g., an anti-NGF antagonistantibody) is identified by incubating an candidate agent with NGF andmonitoring binding and/or attendant reduction or neutralization of abiological activity of NGF. The binding assay may be performed withpurified NGF polypeptide(s), or with cells naturally expressing, ortransfected to express, NGF polypeptide(s). In one embodiment, thebinding assay is a competitive binding assay, where the ability of acandidate agent (such as an antibody) to compete with a known anti-NGFantagonist antibody for NGF binding is evaluated. The assay may beperformed in various formats, including the ELISA format. In otherembodiments, an NGF antagonist (such as anti-NGF antagonist antibody) isidentified by incubating a candidate agent with NGF and monitoringbinding and attendant inhibition of trkA receptor dimerization and/orautophosphorylation.

Following initial identification, the activity of a candidate anti-NGFantagonist (such as an anti-NGF antagonist antibody) can be furtherconfirmed and refined by bioassays, known to test the targetedbiological activities. Alternatively, bioassays can be used to screencandidates directly. For example, NGF promotes a number ofmorphologically recognizable changes in responsive cells. These include,but are not limited to, promoting the differentiation of PC12 cells andenhancing the growth of neurites from these cells (Greene et al., ProcNatl Acad Sci USA. 73(7):2424-8, 1976), promoting neurite outgrowth fromexplants of responsive sensory and sympathetic ganglia (Levi-Montalcini,R. and Angeletti, P. Nerve growth factor. Physiol. Rev. 48:534-569,1968) and promoting the survival of NGF dependent neurons such asembryonic dorsal root ganglion, trigeminal ganglion, or sympatheticganglion neurons (e.g., Chun & Patterson, Dev. Biol. 75:705-711, (1977);Buchman & Davies, Development 118:989-1001 (1993). Thus, the assay forinhibition of NGF biological activity entail culturing NGF responsivecells with NGF plus an analyte, such as a candidate NGF antagonist(including anti-NGF antagonist antibody). After an appropriate time thecell response will be assayed (cell differentiation, neurite outgrowthor cell survival).

The ability of a candidate NGF antagonist (including anti-NGF antagonistantibody) to block or neutralize a biological activity of NGF can alsobe assessed by monitoring the ability of the candidate agent to inhibitNGF mediated survival in the embryonic rat dorsal root ganglia survivalbioassay as described in Hongo et al., Hybridoma 19:215-227 (2000).

Compositions for Use in the Methods of the Invention

The compositions used in the methods of the invention (pertaining torheumatoid arthritis pain and osteoarthritis pain) comprise an effectiveamount of an NGF antagonist (such as an anti-NGF antibody), and, in someembodiments, further comprise a pharmaceutically acceptable excipient.In some embodiments, the composition is for use in any of the methodsdescribed herein. Examples of such compositions, as well as how toformulate, are also described in an earlier section and below. In oneembodiment, the composition comprises an NGF antagonist. In anotherembodiment, the composition comprises one or more NGF antagonists. Inanother embodiment, the composition comprises one or more NGFantagonists selected from any one or more of the following: anantagonist (e.g., an antibody) that binds (physically interacts with)NGF, an antagonist that binds to an NGF receptor (such as a TrkA and/orp75 receptor), and an antagonist that reduces (impedes and/or blocks)downstream NGF receptor signaling. In still other embodiments, thecomposition comprises any NGF antagonist that is not a TrkAimmunoadhesin (i.e., is other than a TrkA immunoadhesin). In otherembodiments, the composition comprises any NGF antagonist that is otherthan an anti-NGF antibody. In still other embodiments, the compositioncomprises any NGF antagonist that is other than a TrkA immunoadhesin andother than an anti-NGF antibody. In other embodiments, an NGF antagonistinhibits (reduces) NGF synthesis, production or release. In someembodiments, the NGF antagonist binds NGF and does not significantlycross-react with related neurotrophins (such as NT3, NT4/5, and/orBDNF). In some embodiments, the NGF antagonist is not associated with anadverse immune response. In some embodiments, the NGF antagonist isselected from the group consisting of an anti-NGF antibody, ananti-sense molecule directed to an NGF (including an anti-sense moleculedirected to a nucleic acid encoding NGF), an anti-sense moleculedirected to an NGF receptor (such as TrkA and/or p75), an NGF inhibitorycompound, an NGF structural analog, a dominant-negative mutation of aTrkA receptor that binds an NGF, a TrkA immunoadhesin, an anti-TrkAantibody, an anti-p75 antibody and a kinase inhibitor. In anotherembodiment, the NGF antagonist is an anti-NGF antibody. In otherembodiments, the anti-NGF antibody recognizes human NGF. In someembodiments, the anti-NGF antibody is human. In still other embodiments,the anti-NGF antibody is humanized (such as antibody E3 describedherein). In still other embodiment, the anti-NGF antibody comprises aconstant region that does not trigger an unwanted or undesirable immuneresponse, such as antibody-mediated lysis or ADCC. In other embodiments,the anti-NGF antibody comprises one or more CDR(s) of antibody E3 (suchas one, two, three, four, five, or, in some embodiments, all six CDRsfrom E3).

It is understood that the compositions can comprise more than one NGFantagonist. For example, a composition can comprise more than one memberof a class of NGF antagonist (e.g., a mixture of anti-NGF antibodiesthat recognize different epitopes of NGF), as well as members ofdifferent classes of NGF antagonists (e.g., an anti-NGF antibody and anNGF inhibitory compound). Other exemplary compositions comprise morethan one anti-NGF antibodies that recognize the same epitope(s),different species of anti-NGF antibodies that bind to different epitopesof NGF, or different NGF inhibitory compounds.

The composition used in the present invention can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and Practice of Pharmacy 20th Ed. (2000)Lippincott Williams and Wilkins, Ed. K. E. Hoover.), in the form oflyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable excipients are further described herein.

The NGF antagonist and compositions thereof can also be used inconjunction with other agents that serve to enhance and/or complementthe effectiveness of the agents. For osteoarthritis pain, NGF antagonistmay be administered in conjunction with one or more other analgesics,NSAIDS, or steroids. Analgesics include, but are not limited to,acetaminophen, tramadol, capsaicin (topical). Examples of NSAIDS areacetylated salicylates including aspirin; nonacetylated salicylatesincluding salsalate, diflunisal; acetic acids including etodolac,diclofenac, indomethacin, ketorolac, nabumetone; propionic acidsincluding fenoprofen, flurbiprofen, ibuprofen, ketoprofen, naproxen,naproxen sodium, oxaprozin; fenamates including meclofenamate, mefenamicacid; phenylbutazone, piroxicam; COX-2 inhibitors including celecoxib,etoricoxib, valdecoxib, rofecoxib, lumiracoxib. An example of steroidsis intraarticular corticosteroids (IACs).

For treating rheumatoid arthritis pain, NGF antagonist may beadministered in conjunction with one or more other analgesics, NSAIDS,corticosteroids (e.g., prednisone), or other disease modifyinganti-rheumatic drugs. Examples of disease modifying anti-rheumatic drugsare methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, TNFinhibitors, soluble interleukin-1 receptor, gold-conjugates, cytotoxicagents (azathiprine, cyclophosphamide, cyclosporine A).

Administration of an NGF Antagonist (Such as an Anti-NGF AntagonistAntibody)

The NGF antagonist (such as anti-NGF antagonist antibody) can beadministered to an individual (for rheumatoid arthritis andosteoarthritis) via any suitable route. It should be apparent to aperson skilled in the art that the examples described herein are notintended to be limiting but to be illustrative of the techniquesavailable. Accordingly, in some embodiments, the NGF antagonist (such asanti-NGF antagonist antibody) is administered to an individual in accordwith known methods, such as intravenous administration, e.g., as a bolusor by continuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,sublingually, intrasynovial, via insufflation, intrathecal, oral,inhalation or topical routes. Administration can be systemic, e.g.,intravenous administration, or localized. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, NGF antagonist (such as anti-NGFantagonist antibody) can be aerosolized using a fluorocarbon formulationand a metered dose inhaler, or inhaled as a lyophilized and milledpowder.

In one embodiment, an NGF antagonist (such as an anti-NGF antagonistantibody) is administered via site-specific or targeted local deliverytechniques. Examples of site-specific or targeted local deliverytechniques include various implantable depot sources of the NGFantagonist (such as anti-NGF antagonist antibody) or local deliverycatheters, such as infusion catheters, an indwelling catheter, or aneedle catheter, synthetic grafts, adventitial wraps, shunts and stentsor other implantable devices, site specific carriers, direct injection,or direct application. See, e.g., PCT Publication No. WO 00/53211 andU.S. Pat. No. 5,981,568.

Various formulations of a NGF antagonist (such as an anti-NGF antagonistantibody) may be used for administration. In some embodiments, the NGFantagonist (e.g., anti-NGF antagonist antibody) may be administeredneat. In some embodiments, NGF antagonist (e.g., anti-NGF antagonistantibody) and a pharmaceutically acceptable excipient may be in variousformulations. Pharmaceutically acceptable excipients are known in theart, and are relatively inert substances that facilitate administrationof a pharmacologically effective substance. For example, an excipientcan give form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing (2000).

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these agents can be combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history.

An anti-NGF antibody can be administered using any suitable method,including by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Anti-NGF antibodies can also beadministered via inhalation, as described herein. Generally, foradministration of anti-NGF antibodies, an initial candidate dosage canbe about 2 mg/kg. For the purpose of the present invention, a typicaldaily dosage might range from about any of 0.1 μg/kg to 1 μg/kg to 3μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg ormore, depending on the factors mentioned above. For example, an anti-NGFantibody may be administered at about 1 μg/kg, about 10 μg/kg, about 20μg/kg, about 50 μg/kg, about 100 μg/kg, about 200 μg/kg, about 500μg/kg, about 1 mg/kg, or about 2 mg/kg. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of symptoms occurs or untilsufficient therapeutic levels are achieved to reduce pain. An exemplarydosing regimen comprises administering an initial dose of about 2 mg/kg,followed by a weekly maintenance dose of about 1 mg/kg of the anti-NGFantibody, or followed by a maintenance dose of about 1 mg/kg every otherweek. However, other dosage regimens may be useful, depending on thepattern of pharmacokinetic decay that the practitioner wishes toachieve. For example, in some embodiments, dosing from one-four times aweek is contemplated. Even less frequent dosing may be used. In someembodiments, the anti-NGF antibody is administered once every 2 weeks,every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 15 weeks,every 20 weeks, every 25 weeks, or longer. In some embodiments, theanti-NGF antibody is administered once every 1 month, every 2 months,every 3 months, every 4 months, every 5 months, every 6 months, orlonger. The progress of this therapy is easily monitored by conventionaltechniques and assays. The dosing regimen (including the NGFantagonist(s) used) can vary over time.

Studies carried out in patients with moderate to severe pain fromosteoarthritis of the knee (summarized in Example 10) demonstrated thatdosages in the range of 3 to 300 μg/kg provided pain relief for varyingdurations. All dosages tested (3, 10, 30, 100, and 300 μg/kg) produced areduction in pain for at least 7 days; higher dosages resulted inprolonged pain relief of at least 28 days (FIG. 24). A dose of 100 μg/kgproduced pain relief for at least 80 days (FIG. 25).

In general, when it is not an antibody, an NGF antagonist may (in someembodiments) be administered at the rate of about 0.1 to 300 mg/kg ofthe weight of the patient divided into one to three doses, or asdisclosed herein. In some embodiments, for an adult patient of normalweight, doses ranging from about 0.3 to 5.00 mg/kg may be administered.The particular dosage regimen, i.e., dose, timing and repetition, willdepend on the particular individual and that individual's medicalhistory, as well as the properties of the individual agents (such as thehalf-life of the agent, and other considerations well known in the art).

For the purpose of the present invention, the appropriate dosage of anNGF antagonist (including an anti-NGF antagonist antibody) will dependon the NGF antagonist (or compositions thereof) employed, the type andseverity of the pain to be treated, whether the agent is administeredfor preventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the agent, and the discretion of theattending physician. Typically the clinician will administer an NGFantagonist (e.g., anti-NGF antagonist antibody), until a dosage isreached that achieves the desired result. Dose and/or frequency can varyover course of treatment.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of pain. Alternatively, sustained continuous releaseformulations of NGF antagonists (e.g., anti-NGF antagonist antibodies)may be appropriate. Various formulations and devices for achievingsustained release are known in the art.

In one embodiment, dosages for an NGF antagonist (e.g., anti-NGFantagonist antibody) may be determined empirically in individuals whohave been given one or more administration(s) of an NGF antagonist.Individuals are given incremental dosages of an NGF antagonist (e.g., ananti-NGF antagonist antibody). To assess efficacy of an NGF antagonist(e.g., anti-NGF antagonist antibody), an indicator of pain can befollowed.

Administration of an NGF antagonist (e.g., an anti-NGF antagonistantibody) in accordance with the method in the present invention can becontinuous or intermittent, depending, for example, upon the recipient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic, and other factors known to skilledpractitioners. The administration of an NGF antagonist (e.g., ananti-NGF antagonist antibody) may be essentially continuous over apreselected period of time or may be in a series of spaced dose, e.g.,either before, during, or after developing pain; before; during; beforeand after; during and after; before and during; or before, during, andafter developing pain.

In some embodiments, more than one NGF antagonist, such as an anti-NGFantagonist antibody, may be present. At least one, at least two, atleast three, at least four, at least five different, or more NGFantagonist (e.g., anti-NGF antagonist antibodies) can be present.Generally, those NGF antagonists (such as anti-NGF antagonistantibodies) have complementary activities that do not adversely affecteach other. NGF antagonists can also be used in conjunction with otheragents that serve to enhance and/or complement the effectiveness of theagents.

Therapeutic formulations of the NGF antagonist (e.g., anti-NGFantagonist antibody) used in accordance with the present invention areprepared for storage by mixing an NGF antagonist (e.g., an antibody)having the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington, The Scienceand Practice of Pharmacy 20th Ed. Mack Publishing (2000)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and may comprise buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Liposomes containing the NGF antagonist (such as anti-NGF antagonistantibody) are prepared by methods known in the art, such as described inEpstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, etal., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time aredisclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes canbe generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing(2000).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antagonist (such as the antibody),which matrices are in the form of shaped articles, e.g. films, ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or ‘poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes. Therapeutic NGF antagonist (e.g., anti-NGFantagonist antibody) compositions are generally placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0.lm, particularly 0.1 and 0.5.lm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an NGFantagonist (such as a nerve growth factor antibody) with Intralipid™. orthe components thereof (soybean oil, egg phospholipids, glycerol andwater).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Treatment efficacy can be assessed by methods well-known in the art.

Kits Comprising Antibodies and Polynucleotides of the Invention

The invention also provides kits comprising antibodies or polypeptidesfor use in detection and/or therapy. Accordingly, in some embodiments,the kits comprise an antibody E3. In some embodiments, the kit comprisesany antibody or polypeptide described herein.

In other aspects, the kits may be used for any of the methods describedherein, including, for example, to treat an individual with pain(including post-surgical pain, rheumatoid arthritis pain, andosteoarthritis pain). The kits of this invention are in suitablepackaging, and may optionally provide additional components such as,buffers and instructions for use of the antibody in any of the methodsdescribed herein. In some embodiments, the kits include instructions fortreating pain. In some embodiments, the kit comprises an anti-NGFantagonist antibody described herein and instructions for treatingand/or preventing rheumatoid arthritis pain in an individual. In otherembodiments, the kit comprises an NGF antagonist (such as an anti-NGFantagonist antibody) described herein and instructions for treatingand/or preventing osteoarthritis pain in an individual. In some of theembodiments, the anti-NGF antagonist antibody is antibody E3.

In another aspect, the invention provides kits comprising apolynucleotide encoding an E3 polypeptide as described herein. In someembodiments, the kits further comprise instructions for use of thepolynucleotide in any of the methods described herein.

Methods for Adjusting the Affinity of an Antibody and Methods forCharacterizing a CDR

We have developed a novel method for characterizing a CDR of an antibodyand/or altering (such as improving) the binding affinity of apolypeptide, such as an antibody, termed “library scanning mutagenesis”.Generally, library scanning mutagenesis works as follows. One or moreamino acid positions in the CDR are replaced with two or more (such as3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)amino acids using art recognized methods. This generates small librariesof clones (in some embodiments, one for every amino acid position thatis analyzed), each with a complexity of two or more members (if two ormore amino acids are substituted at every position). Generally, thelibrary also includes a clone comprising the native (unsubstituted)amino acid. A small number of clones, e.g., about 20-80 clones(depending on the complexity of the library), from each library arescreened for binding affinity to the target polypeptide, and candidateswith increased, the same, decreased or no binding are identified.Methods for determining binding affinity are well-known in the art. Insome embodiments, binding affinity is determined using BIAcore surfaceplasmon resonance analysis, which detects differences in bindingaffinity of about 2-fold or greater. BIAcore is particularly useful whenthe starting antibody already binds with a relatively high affinity, forexample a K_(D) of about 10 nM or lower. Screening using BIAcore surfaceplasmon resonance is described in the Examples, herein.

In other embodiments, binding affinity is determined using KinexaBiocensor, scintillation proximity assays, ELISA, ORIGEN immunoassay(IGEN), fluorescence quenching, fluorescence transfer, and/or yeastdisplay. In other embodiments, binding affinity is screened using asuitable bioassay.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position).

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, in some embodiments, the library comprises substitutions intwo or more positions in one CDR. In other embodiments, the librarycomprises substitution in two or more positions in two or more CDRs. Instill other embodiments, the library comprises substitution in 3, 4, 5,or more positions, said positions found in two, three, four, five or sixCDRs. In some embodiments, the substitution is prepared using lowredundancy codons. See, e.g., Table 2 of Balint et al; (1993) Gene137(1):109-18).

In some embodiments, the CDR is CDRH3 and/or CDRL3. In otherembodiments, the CDR is one or more of CDRL1, CDRL2, CDRL3, CDRH1,CDRH2, and/or CDRH3. In some embodiments, the CDR is a Kabat CDR, aChothia CDR, or an extended CDR.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). For example, as demonstrated in Example 1,use of this method permitted identification of a single substitutionwhich improved binding, even when an estimated 18 other substitutions atthe same amino acid position resulted in no binding (i.e., loss ofantibody function). Candidates that bind may also be sequenced, therebyidentifying a CDR substitution which retains binding.

In some embodiments, multiple rounds of screening are conducted. Forexample, candidates (each comprising an amino acid substitution at oneor more position of one or more CDR) with improved binding are alsouseful for the design of a second library containing at least theoriginal and substituted amino acid at each improved CDR position (i.e.,amino acid position in the CDR at which a substitution mutant showedimproved binding). Preparation, and screening or selection of thislibrary is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acid (including all 20 aminoacids), and positions in the CDRs which cannot be changed or which canonly be changed to a few amino acids. This aspect is discussed andexemplified in Example 1.

In some embodiments, candidates with improved affinity are combined in asecond library, which includes the improved amino acid, the originalamino acid at that position, and may further include additionalsubstitutions at that position, depending on the complexity of thelibrary that is desired, or permitted using the desired screening orselection method. In addition, if desired, adjacent amino acid positioncan be randomized to at least two or more amino acids. Randomization ofadjacent amino acids may permit additional conformational flexibility inthe mutant CDR, which may in turn, permit or facilitate the introductionof a larger number of improving mutations. In some embodiments, thelibrary also comprises substitution at positions that did not showimproved affinity in the first round of screening.

The second library is screened or selected for library members withimproved and/or altered binding affinity using any method known in theart, including screening using BIAcore surface plasmon resonanceanalysis, and selection using any method known in the art for selection,including phage display, yeast display, and ribosome display.

Advantages of the Methods for Adjusting the Affinity of an Antibody andCharacterizing a CDR

The methods are useful for pre-screening CDR amino acid positions inorder to identify amino acid substitutions that improve binding orretain binding. Pre-identification of important residues, substitutionthat improve binding and/or substitutions that retain antibody functionpermits efficient design and screening of an affinity maturationlibrary.

The present method is also useful for characterizing a CDR, and providescomprehensive information regarding the importance of each amino acidposition in a CDR for binding to antigen. The present method may also beused to identify substitutions that improve binding.

The use of small libraries, in which each position may be randomized (insome embodiments, one at a time), permits screening of substitutionmutants using sensitive methods such as BIAcore which provide detailedkinetic information. Screening methods are generally impractical whenlarger libraries are screened. Instead, selection methods, such as phagedisplay, yeast display, and ribosome display, are commonly used toidentify clones that retain binding. Phage display and ELISA assays maydepend heavily on the concentration of the protein sample prepared fromthe clone, and thus tend to be heavily biased towards clones that haveincreased expression, increased stability, or decreased toxicity, ratherthan identifying clones with increased binding affinity. In addition,differences in expression level of the clones may mask smallimprovements in binding affinity. These disadvantages are particularlyacute when an antibody with high binding affinity is used as thestarting material, because very low levels of antigen must be used inorder for screening to be sufficiently stringent.

By contrast, the methods of the invention, such as randomization at eachposition (in some embodiments, one position at a time), permitsintroduction and characterization of the effect of the substitution of,for example, all 20 amino acids at a given position. This analysisprovides information as to how many substitutions at a given positionare tolerated (i.e., retain antibody binding), which in turn, providesinformation relating to the importance of each amino acid for antibodyfunction. Further, substitutions that result in improved binding can beidentified, even under circumstances in which many or most of thesubstitutions at a given position yield non-functional (non-binding)antibodies. By contrast, alanine-scanning mutagenesis, which is commonlyused to identify important CDR positions, provides information relatingto whether the substitution of alanine permits or prevents binding.Generally, positions at which an alanine substitution prevents bindingare removed from the affinity maturation library. In many cases,however, alanine may be a poor substitute at the CDR position.

The present methods also permit identification and characterization ofthe effect of single CDR mutations. By contrast, methods such as phagedisplay introduce and select many mutations simultaneously, and thuspotentially increase the risk that positive mutations will be masked bythe presence of a detrimental mutation present in a particular clone.

The present methods are also useful for improving affinity whileretaining the binding specificity of the original (starting) antibody,insofar as the present methods permit identification of small numbers ofmutations (e.g., 1, 2, 3, 4, or 5 mutations in a single CDR) that resultin improved binding affinity. By contrast, methods such as phage displaytypically improve binding affinity using multiple mutations at once,which may result in shifting specificity of the antibody and/orincreasing undesirable cross-reactivity.

The following examples are provided to illustrate, but not to limit, theinvention.

EXAMPLES Example 1: Humanization and Affinity Maturation of MouseAntagonist Anti-NGF Antibody 911

A. General Methods

The following general methods were used in this example.

Library Generation

Libraries were generated by PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofpeptides and proteins: a laboratory manual, San Diego, Academic Press(see, pages pg 277-291). The doping codon NNK was used to randomize oneamino acid position to include 20 possible amino acids. To randomize oneamino acid position to include only a subset of amino acids withspecific properties, doping codons were used as described in Balint etal, (1993) Gene 137(1):109-18). Site directed mutagenesis was performedusing recombinant PCR as described in Innis et al, (1990) PCR protocols:A guide to methods and applications (see, pp. 177-183).

Small Scale Fab Preparation

Small scale expression in 96 wells plates was optimized for screeningFab libraries. Starting from E. coli transformed with a Fab library,colonies were picked to inoculate both a master plate (agarLB+Ampicillin (50

g/ml)+2% Glucose) and a working plate (2 ml/well, 96 well/platecontaining 1.5 mL of LB+Ampicillin (50

g/ml)+2% Glucose). Both plates were grown at 30° C. for 8-12 hours. Themaster plate was stored at 4° C. and the cells from the working platewere pelleted at 5000 rpm and resuspended with 1 mL of LB+Ampicillin (50

g/ml)+1 mM IPTG to induce expression of Fabs. Cells were harvested bycentrifugation after 5 h expression time at 30° C., then resuspended in500

L of buffer HBS-EP (100 mM HEPES buffer pH 7.4, 150 mM NaCl, 0.005% P20,3 mM EDTA). Lysis of HBS-EP resuspended cells was attained by one cycleof freezing (−80° C.) then thawing at 37° C. Cell lysates werecentrifuged at 5000 rpm for 30 min to separate cell debris fromsupernatants containing Fabs. The supernatants were then injected intothe BIAcore plasmon resonance apparatus to obtain affinity informationfor each Fab. Clones expressing Fabs were rescued from the master plateto sequence the DNA and for large scale Fab production and detailedcharacterization as described below.

Large Scale Fab preparation

To obtain detailed kinetic parameters, Fabs were expressed and purifiedfrom large cultures. Erlenmeyer flasks containing 200 mL ofLB+Ampicillin (50

g/ml)+2% Glucose were inoculated with 5 mL of over night culture from aselected Fab-expressing E. coli clone. Clones were incubated at 30° C.until an OD_(550 nm) of 1.0 was attained and then induced by replacingthe media for 200 ml, of LB+Ampicillin (50

g/ml)+1 mM IPTG. After 5 h expression time at 30° C., cells werepelleted by centrifugation, then resuspended in 10 mL PBS (pH 8). Lysisof the cells was obtained by two cycles of freeze/thaw (at −80° C. and37° C., respectively). Supernatant of the cell lysates were loaded ontoNi-NTA superflow sepharose (Qiagen, Valencia. Calif.) columnsequilibrated with PBS, pH 8, then washed with 5 column volumes of PBS,pH 8. Individual Fabs eluted in different fractions with PBS (pH 8)+300mM Imidazol. Fractions containing Fabs were pooled and dialized in PBS,then quantified by ELISA prior to affinity characterization.

Full Antibody Preparation

For expression of full antibodies, heavy and light chain variableregions were cloned in 2 mammalian expression vectors (Eb.911.E3 orEb.pur.911.3E for light chain and db.911.3E for heavy chain; describedherein) and transfected using lipofectamine into HEK 293 cells fortransient expression. Antibodies were purified using protein A usingstandard methods.

Biacore Assay

Affinities of anti-NGF Fabs and monoclonal antibodies were determinedusing the BIAcore3000™ surface plasmon resonance (SPR) system (BIAcore,INC, Piscaway N.J.). CM5 chips were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiinide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Human NGF was diluted into 10 mM sodium acetate pH 4.0 and injected overthe activated chip at a concentration of 0.005 mg/mL. Using variableflow time across the individual chip channels, two ranges of antigendensity were achieved: 100-200 response units (RU) for detailed kineticstudies and 500-600 RU for screening assays. The chip was blocked withethanolamine. Regeneration studies showed that a mixture of Pierceelution buffer (Product No. 21004, Pierce Biotechnology, Rockford, Ill.)and 4 M NaCl (2:1) effectively removed the bound Fab while keeping theactivity of hNGF on the chip for over 200 injections. HBS-EP buffer(0.01M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% Surfactant P29) wasused as running buffer for all the BIAcore assays.

Screening Assay

A screening BIAcore assay was optimized to determine the affinity of Fabclones from libraries. Supernatants of small culture lysates wereinjected at 50

l/min for 2 min. Dissociation times of 10 to 15 minutes were used fordetermination of a single exponential dissociation rate (k_(off)) usingBIAevaluation software. Samples that showed k_(off) rates in the samerange as the template used to create the library (clone 8L2-6D5, k_(off)1×10⁻³ s⁻¹) were injected for confirmation and dissociation times of upto 45 min were allowed to obtain better k_(off) values. Clones showingimproved (slower) k_(off) values were expressed at large scale and fullkinetic parameters, k_(on) and k_(off), were determined on purifiedprotein. The assay was capable of detecting differences in affinity thatwere approximately 2-fold or larger.

Affinity Determination Assay

Serial dilutions (0.1-10× estimated K_(D)) of purified Fab samples wereinjected for 1 min at 100

L/min and dissociation times of up to 2 h were allowed. Theconcentrations of the Fab proteins were determined by ELISA and/orSDS-PAGE electrophoresis using as a standard a Fab of knownconcentration (as determined by amino acid analysis). Kineticassociation rates (k_(on)) and dissociation rates (k_(off)) wereobtained simultaneously by fitting the data to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values were calculated as k_(off)/k_(on).

B. Humanization and Affinity Maturation of Mouse Antagonist Anti-NGFAntibody 911

The mouse antagonist anti-NGF antibody, 911 (see Hongo et al, (2000)Hybridoma 19(3):215-227) was selected for humanization and affinitymaturation. Mab 911 binds human and rat NGF with high affinity andexhibits no significant cross-reactivity with the neurotrophins NT3,NT4/5 or BDNF. See Hongo, id. The affinity of the papain-cleaved Fabfragment of mouse Mab 911 was determined using BIAcore analysis asdescribed above. The papain-cleaved Fab fragment of mouse Mab 911 boundhuman NGF with a K_(D) of approximately 10 nM.

Humanization and affinity maturation was conducted in several steps, asfollows:

(1) Preparation of CDR-grafted template. The light chain extended CDRsof antibody 911 (i.e., including both the Kabat and Chothia CDR regions)were grafted into the human germline acceptor sequences 08 with JK2 andthe heavy chain extended CDRs of antibody 911 were grafted in to humangermline acceptor sequence VH4-59 with JH4. The amino acid sequences ofthe human germline acceptor sequences are shown in FIGS. 1A and 1B.Amino acid numbering is sequential. Using the protein frameworks notedabove, DNA sequences were designed for synthetic genes encoding humanframework with the murine CDRs. These humanized heavy and light variabledomains were termed hVH and hVL respectively. Codons were optimized forE. coli and hamster usage. Several overlapping oligonucleotides (69-90bases in length) extending the full length of the hVL and hVH with twoshort flanking primers for each chain were used to separately synthesizethe two genes by recursive PCR essentially as described in Prodromou etal, (1992) Protein Eng 5(8): 827-9. Resulting DNA fragments of thecorrect length were gel purified and then cloned into an E. colibicistronic expression plasmid (ampicillin resistant). Expression of theantibodies was under control of an IPTG inducible lacZ promoter similarto that described in Barbas (2001) Phage display: a laboratory manual,Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press (seeVector pComb3X, at pg 2.10), however, modifications included additionand expression of the following additional domains: the human Kappalight chain constant domain (see GenBank Accession No. CAA09181) and theCHI constant domain of IgG2a human immunoglobulin (GenBank Accession No.P01859).

The amino acid sequences of the variable regions of the CDR-graftedantibody (also termed the “template”), termed 8L2-4D5, are also shown inFIGS. 1A and 1B. The affinity of 8L2-4D5 was determined using BIAcoreanalysis as described above. 8L2-4D5 bound human NGF with a K_(D) ofapproximately 38 nM.

(2) Introduction of a point mutation into the framework sequence. TheV71K substitution was introduced into the CDR-grafted heavy chain usingrecombinant PCR site directed mutagenesis as described in Innis et al,(1995) PCR strategies, San Diego, Academic Press. This substitutionreplaced the human framework residue with the corresponding mouseframework residue. The resulting antibody was termed 8L2-6D5, and theamino acid sequence of the heavy chain variable region of 8L2-6D5 isshown in FIG. 1A. The affinity of 8L2-6D5 was determined using BIAcoreanalysis as described above. The Fab fragment of 8L2-6D5 bound human NGFwith a Kd of approximately 15 nM. 8L2-6D5 was chosen as template foraffinity maturation.

(3) Humanization and affinity maturation of CDRs L1, L2, H1 and H2. CDRsL1, L2, H1 and H2 were subjected to humanization and affinitymaturation. Amino acid positions in CDRs L1, L2, H1, and H2 wereidentified that are not essential for the structure of the CDRs based onthe Chothia canonical structure (see Al-Lazikani et al (1997) J. Mol.Biol. 273(4):927-48); and subjected to randomization as follows. Twolibraries were prepared containing the light chain mutations or heavychain mutations shown in Table 2, and the grafted (mouse) CDR L3 or CDRH3, respectively, using PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofpeptides and proteins: a laboratory manual, San Diego, Academic Press,using doping codons as described in Balint et al, (1993) Gene137(1):109-18). Generally, the amino acid residues were altered toresidues that are more common in human antibodies, based on alignmentsof antibody 911 light chain and heavy chain amino acid sequences withhuman germline antibody sequences. The wildtype (unsubstituted) aminoacid residue was also represented in the library with the exception ofCDR H2 residue 50, a methionine, in which the wildtype methionine wasnot represented in the library. Methionine residues are subject tooxidation; thus, replacement of that residue was expected to improvestability of the resulting antibody. The libraries of Fabs were clonedinto vector pComb3X plus the human CH1 and Cκ regions, as describedabove.

TABLE 2 1. Heavy chain H1/H2 library: CDR-H1 I34 was changed to F, L, V,S, P, T, A, or I N35 was changed to N, T, S, or Y CDR-H2 M50 was changedto all 20 natural amino acids A62 was changed to A or S L63 was changedto L or V 2. Light chain L1/L2 library CDR-L1 S26 was changed to S, A,V, or F D28 was changed to D, A, S, or Y H32 was changed to H, N, K, D,E, Q, or Y CDR-L2 Y50 was changed to Y, D, A, or S I51 was changed to I,T, A, or V F54 was changed to F or L S56 was changed to S and T

For affinity screening experiments, each library was further paired withthe corresponding CDR-grafted light or heavy chain (for example, theH1/H2 library was paired with CDR-grafted light chain), the antibody wasexpressed, and affinity to human NGF of the individual clones wasscreened using the BIACORE surface plasmon resonance (SPR) system(BIAcore, Inc. Piscataway, N.J.) according to the manufacturer'sinstructions and as described above. k_(off), k_(on) and K_(D) weredetermined. Antibody clones were ranked based on k_(off) rates, sincegenerally most variation in affinity is seen in k_(off) rates, andfurther because k_(off) rates are independent of antibody concentration.

The sequence of clones that bound was determined and the sequence ofclones that bound is shown in table 3.

TABLE 3 L1 and L2 amino acid sequences, H1 and H2 aminoacid sequences, and kinetic data for clones thatbound following affinity screening of H1/H2 or L1/L2 library clones.CDR 1-2 mutants kinetic data Light chain library clones Paired with 8L2CDRL1 CDRL2 heavy chain AA sequence AA sequence k_(off) (s−1)*K_(D) (nM) 8L2-6D5 RASQDISNHLN YISRFHS **1e−3 25 (control)(SEQ ID NO: 12) (SEQ ID NO: 13) L129 RASQSISNNLN YTSRFHS 4.5e−4 11(SEQ ID NO: 18) (SEQ ID NO: 19) L208 RASQYISNHLN YTSRFHS 4.6e−4 11(SEQ ID NO: 20) (SEQ ID NO: 21) L97 RASQSISNQLN YVSRFHS 5.6e−4 14(SEQ ID NO: 22) (SEQ ID NO: 23) L81 RAFQAISNQLN YISRFHT 7.4e−4 18(SEQ ID NO: 24) (SEQ ID NO: 25) L6 RAFQSISNQLN YASRFHS 8.2e−4 20(SEQ ID NO: 26) (SEQ ID NO: 27) Heavy chain library clonesPaired with 6D5 CDRH1 CDRH2 Light chain AA sequence AA sequencek_(off) (s−1) *K_(D) (nM) 8L2-6D5 GFSLIGYDIN MIWGDGTTDYNSAL   1e−3 25(control) (SEQ ID NO: 9) (SEQ ID NO: 10) H109 GFSLIGYDSN IIWGDGTTDYNSAL1.6e−4 4 (SEQ ID NO: 28) (SEQ ID NO: 29) H19 GFSLIGYDLN IIWGDGTTDYNSAV2.4e−4 6 (SEQ ID NO: 30) (SEQ ID NO: 31) H222 GFSLIGYDVT GIWGDGTTDYNSAV3.8e−4 9.5 (SEQ ID NO: 32) (SEQ ID NO: 33) H225 GFSLIGYDVTGIWGDGTTDYNSSV 3.8e−4 9.5 (SEQ ID NO: 34) (SEQ ID NO: 35) H18 GFSLIGYDATGIWGDGTTDYNSAV 4.2e−4 10.5 (SEQ ID NO: 36) (SEQ ID NO: 37) H9 GFSLIGYDVSIIWGDGTTDYNSSV 4.1e−4 10.2 (SEQ ID NO: 38) (SEQ ID NO: 39) H227GFSLIGYDIS QIWGDGTTDYNSSV 5.4e−4 13.5 (SEQ ID NO: 40) (SEQ ID NO: 41)H17 GFSLIGYDAS GIWGDGTTDYNSSV 6.1e−4 15.2 (SEQ ID NO: 42)(SEQ ID NO: 43) H28 GFSLIGYDST SIWGDGTTDYNSAL 7.5e−4 18.7(SEQ ID NO: 44) (SEQ ID NO: 45) AA in bold were randomized as indicatedabove *KD calculated using k_(on) 4e4 M⁻¹s⁻¹ **For convenience, “e” asused herein denotes “x10.” Thus, 4e4 interchangeably means 4 x l0⁴.

CDRs containing the following substitutions retained binding:

CDR-H1

-   -   I34: S, L, V, I and A bound.    -   N35: N, T and S bound.

CDR-H2

-   -   M50: M, I, G, Q, S, L bound.    -   A62: A and S bound.    -   L63: Land V bound.

CDR-L1

-   -   S26: S, and F bound.    -   D28: D, S, A, Y bound.    -   H32: H, N, Q bound.

CDR-L2

-   -   Y50: Y bound.    -   I51: I, T, V, A, bound.    -   F54: F bound    -   S56: S and T bound

CDRs containing the following substitutions were selected generallybased on binding affinity and combined into a single clone, termedH19-L129:

CDR-H1: I34L; N35N (no change)

CDR-H2: M50I; A62A (no change); L63V

CDR-L1: S26S (no change); D28S; H32N

CDR-L2: Y50Y (no change); I51T; F54F (no change); S56S (no change)

These mutations were combined (by amplifying the H and L chains by PCR,cutting the PCR products and vector (pRN8) with restriction enzyme andperforming a 3 fragment ligation) into a single clone, termed H19-L129,which also included the grafted H3 and L3 CDRs. The sequence of theheavy chain and light chain variable regions of H19-L129 is shown inFIGS. 1A and 1B, and Table 4 shows the amino acid sequence of CDRs L1,L2, H1 and H2. H19-L129 bound NGF with a KD of approximately 1 nM, asdetermined using BIAcore analysis as described herein.

TABLE 4 Amino acid sequence of CDRs H1, H2, L1 and L2 andkinetic data for combined clone H19-L129.  Combination clone: mutationsCDRL1 CDRL2 in CDRs H1, H2, CDRH1 CDRH2   L1, L2 AA sequence AA sequencek_(off) (s−1) *K_(D) (nM)  H19-L129 CDR-L1: CDRL2: 1.1e−4 3.5 RASQSISNNLN YTSRFHS  (SEQ ID NO: 18) (SEQ ID NO: 19)  CDR H1: CDR-H2: GFSLIGYDLN IIWGDGITDYNSAV  (SEQ ID NO: 30) (SEQ ID NO: 31)  *KDcalculated using k_(on) 4e4 M⁻¹s⁻¹

(4) Affinity maturation of H3 and L3 CDRs. Affinity maturation of the H3and L3 CDRs was carried out in two steps. First, in a process termed“library scanning mutagenesis”, each amino acid residue in H3 and L3 wasindividually prescreened in order to identify amino acid positions atwhich a mutation resulted in increased binding affinity to human NGF.Based on the results of the library scanning mutagenesis (also termed“small library randomization analysis”), a subset of amino acidpositions in H3 and L3 were selected for preparation of the affinitymaturation library, and the affinity maturation library was screened foraffinity to human NGF using BIAcore analysis as described herein. It isappreciated that these techniques can be generally applied.

(a) Library Scanning Mutagenesis

Each amino acid position in the H3 and L3 CDRs was individuallyprescreened for substitutions which resulted in increased bindingaffinity to human NGF. The frequency of amino acid substitutions at anygiven position that resulted in improved binding, the same binding,worse binding or no binding provided information relating to relating topositions in the CDRs that can be changed to many different amino acid(including all 20 amino acids), and positions in the CDRs which cannotbe changed or which can only be changed to a few amino acids. Amino acidsubstitutions resulting in increased binding affinity were alsoidentified. Based on the results of this screening, a subset of aminoacid positions in CDRs H3 and L3 were selected for preparation of anaffinity maturation library.

Individual Fab libraries were prepared in which each amino acid of L3and H3 CDRs was randomized to all 20 amino acids, one at a time,resulting in several (5 libraries for the light chain and 13 librariesfor the heavy chain) small libraries, each with a complexity of 20 aminoacid possibilities at each amino acid position. In all cases, the native(i.e., unchanged) amino acid was represented in the library. Librarieswere prepared by PCR cassette mutagenesis with degenerateoligonucleotides as described in Kay et al. (1996), Phage display ofPeptides and Proteins: a laboratory manual, San Diego, Academic Press,using the doping codon NNK to randomize one amino acid position toinclude 20 possible amino acids. The 8L2-6D5 (the CDR grafted antibody,having the framework mutation V71K) served as the template for libraryconstruction because the lower affinity of the CDR grafted antibodypermitted easier detection of differences in affinity in H3 and L3mutants during screening. Thus, each member of a library contained aCDR3 (either H3 or L3) with one amino acid substitution, and 5 graftedCDRs.

20-80 clones from each small library were screened using BIAcoreanalysis as described herein. Samples were simultaneously analyzed byBIAcore for binding affinity to NGF in one channel of the BIAcore chipand for presence of Fab by binding to a penta-histag antibody in anotherchannel of the sensor chip, to detect the his tag at the C terminus ofthe heavy chain. Clones that expressed protein were classified as havingthe same affinity, worse affinity, better affinity or no binding, usingkoff to classify: The results of this analysis are shown in Table 5.

TABLE 5 Clones that expressed protein were classified as having the sameaffinity, worse affinity, better affinity or no binding, based on koff.Percentage of AAs that same retain better >le-3, Worse no bindingmutation 1e−3< 2e−3< ≥2e−3 bind capacity Light chain L_S91X 13% 40% 20%26% 50% L_K92X 100%  ~100%    L_T93X 93%  7% 93% L_L94X 40% 60% 40%L_Y96X 13% 80%  7% 13% Heavy chain H_G98X 50% 37% 13% 50% H_G99X 46% 54%46% H Y100X 26% 73% 26% H Y101X  6% 12% 82%  6% H_Y102X  7% 25    68% 7% H_G103X  4% 21% 16% 58% 25% H_T104X 20% 30% 50% 20% H_S105X 10% 25%26% 39% 35% H_Y106X 75% 25% 75% H_Y107X  8% 46% 46%  8% H_F108X 23% 27%50% 23% H_D109X 29% 46% 25% 29% H_Y110X 90%  5%  5% 90%

The sequence of all clones with improved affinity was determined,revealing the frequency and identity of amino acid substitutions thatresulted in increased affinity. In addition, a few clones that retainedan affinity similar to the 812-6D5 clone were selected from eachlibrary, in order to ascertain amino acid sequence substitutions thatwere permitted at a given position, even though the substitution did notnecessarily increase binding affinity. The results of this analysis aresummarized in Table 6.

TABLE 6 CDR H3 mutations (8L2-6D5 template, including antibody 911CDR-H3 amino acid sequence: GGYYYGTSYYFDY k_(off) (s−1) K_(D)* (nM) (SEQID NO: 11) 1E−3 25 Y100L  1.2E−3 30 Y100R  1.1E−3 27 Y101W  5.6E−4 14G103A  1.6E−4  4 T104S  2.2E−3 55 S105A  5.1E−4 13 S105T  6.4E−4 16Y106R  1.6E−3 40 Y106T  2.0E−3 50 Y106M  2.7E−3 67 Y107F  1.4E−3 35F108W 1.22E−3 30 D109N  1.5E−3 37 D109G   1E−3 25 Y110K  1.4E−3 35 Y110S 1.5E−3 37 Y110R  1.6E−3 40 Y110T  1.7E−3 42 CDR L3 mutations (8L2-6D5template, including wildtype (unsubstituted) CDR-L3 amino acid sequence:QQSKTLPYT k_(off) (s−1) K_(D)* (nM) (SEQ ID NO:_14) 1E-3 25 S91E  2.5E-4 6 Y96R  1.7E-3 42 *KD calculated using k_(on) 4e4 M⁻¹s⁻¹

Several mutations resulted in increased binding affinity. At least thefollowing mutations resulted in significantly increased binding affinityas compared with the 8L2-6D5 template: (H_Y101W (CDR sequenceGGYWYGTSYYFDY (SEQ ID NO:46)); H_S105A (CDR sequence GGYYYGTAYYFDY (SEQID NO:47)); H_S105T (CDR sequence GGYYYGTTYYFDY (SEQ ID NO:48)); H_G103A(CDR sequence GGYYYATSYYFDY (SEQ ID NO:49); and L_S91E (CDR sequenceQQEKTLPYT (SEQ ID NO:50)).

The results of this experiment were used to guide selection of aminoacid positions for generation of the affinity maturation libraries.

This experiment also provided information regarding the frequency ofamino acid substitutions at any given position that resulted in improvedbinding, the same binding, worse binding or no binding, as shown inTable 5. This information permitted identification of amino acidpositions in the CDRs that could be changed to many different amino acid(including all 20 amino acids), and positions in the CDRs which could bechanged to a few amino acids or a very few amino acids (in someembodiments, no amino acids). These results also demonstrated amino acidsubstitutions that increased binding affinity.

(b) Affinity Maturation

Next, the results of the small library randomization analysis (above)were used to select residues for production of the H3 and L3 librariesfor affinity maturation of the H3 and L3 CDRs. Residues Y101 and G103 ofCDR H3 and residues S91 and K92 of CDR L3 were selected for productionof the H3 and L3 libraries for affinity maturation of the H3 and L3CDRs.

This library combined mutations in H3 and L3 at the same time inCDR-grafted clone 8L2-6D5, and separately in the background of H19-L129,and had a diversity of 80 different clones. Table 7 shows the amino acidresidues selected for substitution and the amino acids that weresubstituted at each position.

TABLE 7 Amino acid residues in H3 and L3 selected for substitution andthe amino acids that were substituted at each position CDR-H3: Y101 waschanged to Y and W, C. (Note that C was included because use of codonTRS in one degenerated oligonucleotide also generated codon C). G103 waschanged to A, P, S CDR-L3: S91 was changed to E. K92 was changed to alltwenty amino acids. A, R, K, and H bound.

Each polypeptide was expressed as a Fab, and affinity to human NGF of 96individual clones was screened for each library using BIACORE analysisaccording to the manufacturer's instructions and described above. Theresults of this analysis are shown in Table 8.

TABLE 8 CDR L3 H3 COMBINATION mutations k_(off) (s−1) K_(D)* (nM)(8L2-6D5 template) 1E−3 25 L_S91E; L_K92A 5.5E−4 13 (CDR sequenceQQEATLPYT (SEQ ID NO: 51)) H_Y101W; H_G103A (CDR sequence GGYWYATSYYFDY(SEQ ID NO: 52)) L_S91E; L_K92R 1.0E−4 25 (CDR sequence QQERTLPYT (SEQID NO: 53)) H_Y101W; H_G103A (CDR sequence GGYWYATSYYFDY (SEQ ID NO:54)) CDR L3 H3 COMBINATION mutations k_(off) (s−1) (H19-L129 template,H1H2L1L2 matured) 1.1e−4 K_(D)* (nM) L_S91E; L_K92H 1.2E−5  0.3 (CDRsequence QQEHTLPYT (SEQ ID NO: 55)) H_Y101W; H_G103A (CDR sequenceGGYWYATSYYFDY (SEQ ID NO: 56)) (CLONE E3) L_S91E; L_K92S 4.7E−5  1.1(CDR sequence QQESTLPYT (SEQ ID NO: 57)) H_Y101W; H_G103S (CDR sequenceGGYWYSTSYYFDY (SEQ ID NO: 58)) L_S91E; L_K92K   2E−5  0.5 (CDR sequenceQQEKTLPYT (SEQ ID NO: 59)) H_Y101Y; H_G103A (CDR sequence GGYYYATSYYFDY(SEQ ID NO: 60)) L_S91E; L_K92R 1.4E−5  0.35 (CDR sequence QQERTLPYT(SEQ ID NO: 61)) H_Y101W; H_G103A (CDR sequence GGYWYATSYYFDY (SEQ IDNO: 62)) (CLONE 3C) L_S91E; L_K92R 1.5E−5  0.37 (CDR sequence QQERTLPYT(SEQ ID NO: 63)) H_Y101Y; H_G103A (CDR sequence GGYYYATSYYFDY (SEQ IDNO: 64)) *KD calculated using k_(on) 4e4 M⁻¹s⁻¹

Based on binding affinity, the best clones, E3 (interchangeably termed“3E”) and 3C, were selected for further characterization. E3 comprisedthe following CDR substitutions: CDR-H3: Y101W, G103A; and CDR-L3: S91E,K92H, which were combined into a single clone which also included thefollowing L1, L2, H1 and H2 mutations:

CDR-H1: I34L;

CDR-H2: M50I; L63V;

CDR-L1: D28S; H32N;

CDR-L2: I51T.

The sequence of the heavy chain and light chain variable regions of E3is shown in FIGS. 1A and 1B. 3C comprised the following CDRsubstitutions: CDR-L3: S91E; K92R; CDRH3: Y101W; G103A, which werecombined into a single clone which also included the L1, L2, H1 and H2mutations described for clone 3E.

3E and 3C sequences were cloned into mammalian expression vectors forproduction of Fab and full antibody, and expressed in HEK293 cells andpurified using Ni-NTA or protein A chromatography. Pure protein wasaccurately quantified by amino acid analysis.

The binding affinities to human NGF of Fabs E3 and 3C were measuredusing BIAcore analysis according to the manufacturer's instructions andas described above, except that 100 RU NGF was used on chip to prevent arebinding effect. Briefly, several concentrations of antibodies (Fabs)were injected for 2 minutes onto a CM5 chip with 100 RU of immobilizedhuman NGF on it, and permitted to dissociate for 1800 seconds. Mouseantibody 911 (Fab) was analyzed as a control. Data was analyzed usingBIAevaluation software following the manufacturer's instructions. Theresults of the analysis of antibody E3 and 911 are shown in FIGS. 9 and10. E3 bound human NGF with a KD of approximately 0.07 nM (and with akon of about 6.0e5 M-1s-1, and a k_(off) of about 4.2e-5 s-1). 3C boundhuman NGF with a KD of approximately 0.35 nM (with a k_(off) of about1.4E-5). By contrast, mouse antibody 911 bound NGF with a KD of 3.7 nM,k_(off) of 8.4×10⁻⁵s¹ and k_(on) of 2.2×10⁴ Ms⁻¹.

Antibody E3 (interchangeably termed 3E) was selected for furtheranalysis based on the high binding affinity. To test the ability of E3to prevent the interaction of NGF with the NGF receptors trkA and p75,2.5 nM of human NGF was premixed and incubated for one hour with 0 to 50nM of antibody E3 (Fab). After the incubation, samples were injected at10 ul/minute on a BIAcore CM5 chip containing 260 RU of p75 (channel 2)and 600 RU of trkA (channel 3), and percent binding was determined. Theresults of this analysis are shown in FIG. 11. Increased concentrationsof Fab E3 blocked the interaction of NGF with both p75 and trkA, asshown by decreased signal (measured in RU), indicating that Fab E3blocks the interaction of human NGF with both trkA and p75. Whenantibody E3 (Fab) concentration equaled NGF concentration (at about 2.5nM NGF concentration), no NGF binding was observed (as shown by a signalof zero). The fact that zero percent NGF-receptor binding occurred whenconcentration of NGF was equal to antibody 3E concentration suggestedthat 2.5 nM NGF was at least ten-fold higher than the kD of E3 for NGFand at equilibrium.

Example 2: Evaluation of NGF-Blocking Ability of Anti-NGF AntibodiesUsing Mouse E13.5 Trigeminal Neuron Survival Assay

The ability of Fab E3 or full antibody E3 to block NGF activity wasevaluated by measurement of the capacity of the antibody to inhibitNGF-dependent survival of mouse E13.5 trigeminal neurons in vitro. Thetrigeminal ganglion is comprised of cutaneous sensory neurons thatinnervate the facial region. The survival of mouse E13.5 trigeminalneurons is a sensitive assay to evaluate the NGF-blocking activity ofanti-NGF antagonist antibodies because NGF is required to supportsurvival of these neurons. For example, at saturating concentrations ofNGF, the survival is close to 100% by 48 hours in culture. By contrast,less than 5% of the neurons survive by 48 hours in absence of NGF.

The survival assay was conducted as follows: time-mated pregnant SwissWebster female mice were euthanised by CO2 inhalation. The uterine hornswere removed and the embryos at embryonic stage E13.5 were extracted anddecapitated. The trigeminal ganglia were dissected usingelectrolytically sharpened tungsten needles. The ganglia were thentrypsinized, mechanically dissociated and plated at a density of 200-300cells per well in defined, serum-free medium in 96-well plates coatedwith poly-L-ornithine and laminin.

The blocking activity of anti-NGF Fabs or antibodies was assessed byadding to the trigeminal neurons varying doses of anti-NGF antibodiesMab 911 (Fab), 8L2-6D5; H19-L129; E3 and 3C; and human or rat NGF at thefollowing concentrations: 0.4 ng/ml (˜15 pM; this concentrationrepresented a saturating concentration of NGF for survival) and 0.04ng/ml (˜1.5 pM; this concentration is around the IC50). After 48 hoursin culture, the cells were subjected to an automated immunocytochemistryprotocol performed on a Biomek FX liquid handling workstation (BeckmanCoulter) as follows: fixation using 4% formaldehyde, 5% sucrose, andPBS; permeabilization using 0.3% Triton X-100 in PBS); blocking ofunspecific binding sites using 5% normal goat serum, 0.11% BSA in PBS;and sequential incubation with a primary and secondary antibodies todetect neurons. The primary antibody was rabbit polyclonal antibodyagainst the protein gene product 89.5 (PGP9.5, Chemicon), an establishedneuronal phenotypic marker. The secondary antibody was Alexa Fluor 488goat anti-rabbit (Molecular Probes), together with the nuclear dyeHoechst 33342 (Molecular Probes) to label the nuclei of all the cellspresent in the culture. Image acquisition and image analysis wereperformed on a Discovery-I/GenII Imager (Universal Imaging Corporation).Images were automatically acquired at two wavelengths for Alexa Fluor488 and Hoechst 33342, with the nuclear staining being used as referencepoint for the image-based auto-focus system of the Imager, since nuclearstaining is present in all of the wells. Appropriate objectives andnumber of sites imaged per well were selected to cover the entiresurface of each well. Automated image analysis was set up to count thenumber of neurons present in each well after 48 hours in culture basedon their specific staining with the anti-PGP9.5 antibody. Carefulthresholding of the image and application of morphology and fluorescenceintensity based selectivity filter resulted in an accurate count ofneurons per well.

The results of this experiment demonstrated that Fab E3 blocked NGFactivity with a high affinity. The results are shown in FIGS. 4-6, andTable 9.

FIG. 4 is a graph showing NGF-dependent survival of E13.5 neurons in thepresence of varying concentration of human and rat NGF.

FIG. 5 is a graph comparing the NGF blocking effect of various Fabs inthe presence of either 0.04 ng/ml of human NGF (approximately 1.5 pM;shown in the lower panel) or 0.4 ng/ml human NGF (approximately 15 pM;shown in the upper panel). 1.5 pM of NGF was around the EC50 of NGFpromoting survival, while 15 pM represented a saturating concentrationof NGF. Survival of E13.5 mouse trigeminal neurons in variousconcentrations of Fab E3; murine 911 Fab; and Fab H19-L129 and Fab8L2-6D5 was assessed as described above. The IC50 (in pM) was calculatedfor each Fab at each NGF concentration, and is shown in Table 9. Fab E3strongly blocked human NGF-dependent trigeminal neuron survival, with anIC50 of approximately 21 pM in the presence of 15 pM human NGF, and anIC50 of approximately 1.2 pM in the presence of 1.5 pM human NGF. Fabs3C and H19-L129 also strongly blocked human NGF-dependent trigeminalneuron survival.

FIG. 6 is a graph comparing the NGF blocking effect of various Fabs inthe presence of either 0.04 ng/ml of rat NGF (approximately 1.5 pM;shown in the lower panel) or 0.4 ng/ml rat NGF (approximately 15 pM;shown in the upper panel). 1.5 pM of NGF was around the EC50, while 15pM represented a saturating concentration of NGF. Survival of E13.5mouse trigeminal neurons in various concentrations of Fab E3; murine Fab911; and Fab H19-L129 and 8L2-6D5 was assessed as described above. TheEC50 (in pM) was calculated for each Fab at each NGF concentration, andis shown in Table 9. Fab E3 strongly blocked human NGF-dependenttrigeminal neuron survival, with an IC50 of approximately 31.6 pM in thepresence of 15 pM rat NGF, and an IC50 of approximately 1.3 pM in thepresence of 1.5 pM rat NGF. Fabs 3C and H19-L129 also strongly blockedrat NGF-dependent trigeminal neuron survival.

TABLE 9 IC50 (in the presence IC50 (in the presence of 15 pM NGF) of 1.5pM NGF) Human NGF pM pM 8L2-6D5 Fab 1580.5 461.8 H19-L129 Fab 60.1 9.63E Fab <21.0 <1.2 3C Fab 80.9 5.6 911 Fab 322.3 63.5 IC50 (15 pM NGF)IC50 (1.5 pM NGF) Rat NGF pM pM 8L2-6D5 Fab 730.3 169.4 H19-L129 Fab31.0 6.0 3E Fab <8.3 <1.3 3C Fab 31.6 6.0 911 Fab 161.0 34.6

In a different experiment, we compared the ability of full antibody E3and Fab 3E to inhibit NGF-dependent survival of E13.5 neurons in thepresence of 0.4 ng/ml (saturating concentration) of human NGF. Theresults of the analysis are shown in FIG. 12. Full antibody E3 and Fab3E showed similar levels of inhibition of NGF-dependent survival whenthe concentration of whole antibody and Fab were normalized to thenumber of NGF binding sites (Fab has one binding site and whole antibodyhas two binding sites). These results demonstrated that there was noavidity effect due to the binding of a full antibody to the NGF dimer.

In another experiments, we compared the ability of variousconcentrations (20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, and 0.0 nM) ofantibody E3, antibody 911, and a trkA receptor immunadhesin (consistingof the extracellular domain of the NGF receptor trkA fused with theimmunoglobulin Fc domain, CH2-CH3) to inhibit NGF-dependent survival ofE13.5 neurons in the presence of 0.4 ng/ml (saturating conditions).These results are shown in FIG. 13. These results demonstrated thatantibody E3 blocked NGF better than either antibody 911 or the trkAimmunoadhesin.

Example 3: Evaluation of the Specificity of Anti-NGF Antibody E3 UsingMouse Trigeminal and Nodose Neuron Survival Assays

The ability of antibody E3 to specifically block NGF activity wasevaluated by measurement of the capacity of the antibody to inhibitsurvival of mouse E17/18 trigeminal neurons in vitro in the presence ofsaturating concentrations of NGF, the NGF-related neurotrophin NT3, orthe NGF-unrelated neurotrophic factor, macrophage stimulating protein(MSP). The survival of mouse E17/18 trigeminal neurons is a sensitiveassay to evaluate the NGF-blocking activity of anti-NGF antagonistantibodies because NGF is required to support survival of these neuronsat higher concentrations than the level of NGF required to supportsurvival of E13.5 TG neurons). Survival of these neurons is alsosupported by NT3 or MSP; therefore, the survival of these neurons isalso a sensitive assay to evaluate whether the anti-NGF antagonistantibody also blocked NT3 or MSP.

The ability of antibody E3 to specifically block NGF activity was alsoevaluated by measurement of the capacity of the antibody to inhibitsurvival of mouse nodose E17 neurons in the presence of saturatingconcentrations of BDNF or NT4/5. Survival of nodose neurons is supportedby BDNF or NT4/5; therefore, survival of these neurons is a sensitiveassay to evaluate the BDNF or NT4/5-blocking ability of the anti-NGFantagonist antibody.

The survival assay was conducted as follows: time mated pregnant SwissWebster female mice were euthanised by CO2 inhalation. The uterine hornswere removed and the embryos (at embryonic day 17 or 18) were extractedand decapitated. The trigeminal and nodose ganglia were dissected andcleaned. The ganglia were then trypsinised, mechanically dissociated andplated at a density of 100-300 cells per well in defined, serum-freemedium in 4-well plates (Greiner) coated with poly-L-ornithine andlaminin.

E17/18 trigeminal neurons were grown either without added neurotrophicfactors (negative control) or in the presence of saturatingconcentrations of human NGF (400 pM and 15 pM) (positive control); NT3(400 pM); or MSP (600 pM). Duplicate cultures were set up that includedvarying concentrations of E3 and 911 Fabs and full antibodies.Concentration of Fab and full antibodies was indicated per binding site(e.g., a full antibody contains two binding sites, while a Fab containsone binding site).

E17 nodose neurons were grown either in the absence of addedneurotrophic factors (negative control), or with saturatingconcentrations of BDNF (400 pM) (positive control) or NT4/5 (400 pM) orNGF unrelated growth factor TLF (interleukin inhibitory factor). Highconcentrations of neurotrophins were used, as the goal of thisexperiment was to test specificity of the antibodies. Duplicate cultureswere set up that included varying again with and without the addition ofantibodies E3 and 911. After 48 hours in culture the total number ofneurons surviving in each well under each condition was ascertained bymanual counting using a phase-contrast microscope.

The results of these experiments demonstrated that E3 and 911 antibodiescompletely blocked the survival promoting effects of NGF on E18trigeminal neurons. By contrast, E3 and 911 antibodies had no effect onsurvival of trigeminal neurons promoted by NT3 or MSP, or survival ofnodose neurons promoted by BDNF or NT4/5 or LIF. These resultsdemonstrated that antibody E3 possessed selective specificity for NGF,as there was no detected interaction between these antibodies and otherNGF related neurotrophins (NT3, NT4/5, BDNF) at concentrations 1000-foldto 10,000-fold higher than effective concentration for NGF blocking.Further, these results demonstrated that the neuronal death seen inNGF-supplemented cultures of NGF-dependent neurons on addition ofantibody or Fab E3 was due to a specific interaction between theseantibodies and NGF and was not due to a generalized toxic effect. Mouseanti-NGF antagonist antibody 911 was also tested, and similar resultswere observed. Note that due to the high concentrations of neurotrophinsused, both antibody E3 and 911 are very close to their titrationconditions and were expected to bind NGF at similar levels because thedifferences in binding affinity of these antibodies to NGF would to beless apparent under these conditions.

The results of these experiments are shown in FIGS. 14, 15, 16, and 17.The data showed mean percent survival after 48 hours in culture(±standard error of mean, n=3 for each data point) relative to thesurvival seen in the positive control for each experiment (e.g., 100%survival of trigeminal neurons grown in the presence of saturating NGFconcentration, and 100% survival of nodose neurons grown in the presenceof saturating BDNF concentration, respectively). FIGS. 14-15 are graphsshowing that anti-NGF antagonist antibody E3 or Fab E3 did not inhibitthe survival promoted by NT3, and MSP, even at antibody concentrationsas high as 200 nM. By contrast, 20 nM of antibody E3 or Fab 3E and Fab911 totally blocked NGF-elicited survival. Mouse anti-NGF antagonistantibody 911 was also tested, and similar results were observed.Specifically, FIG. 14 is a graph showing comparison of the effect ofvarious concentrations (20 nM, 2 nM, or 0.2 nM) of E3 Fab (termed “3E”in the figure) and mouse antibody 911 Fab on survival of E18 trigeminalneurons in the presence of no added neurotrophin (termed “control”), 400pM NGF (termed “NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM) or 600 pM MSP(termed “MSP-600 pM). FIG. 15 is a graph depicting comparison of theeffect of various concentrations (200 nM and 80 nM) of E3 Fab and fullantibody and mouse antibody 911 full antibody and Fab of survival of E17trigeminal neurons in the presence of no added neurotrophins (termed “nofactor”), 400 pM NGF (termed “NGF-400 pM), 10 nM NT3 (termed “NT3-10 nM)or 600 pM MSP (termed “MSP-600 pM).

FIG. 16-17 are graphs showing that anti-NGF antagonist antibody E3 orFab E3 did not inhibit survival of E17 nodose neurons promoted by BDNF,NT4/5 or LIF. Mouse anti-NGF antagonist antibody 911 was also tested,and similar results were observed. Specifically, FIG. 16 is a graphshowing comparison of the effect of various concentrations (200 nM or 80nM) of full antibody E3 (termed “3E in the figure”), Fab E3, fullantibody 911, or Fab 911 on the survival of E17 nodose neurons in thepresence of no added neurotrophins (termed “no factors”), 400 pM BDNF(termed “BDNF-400 pM), 400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nMLIF (termed “LIP-2.5 nM). FIG. 17 is a graph showing comparison of theeffect of various concentrations (200 nM, 20 nM, 2 nM) of Fab E3 (termed“3E in the figure”), or Fab 911 on the survival of E17 nodose neurons inthe presence of no added neurotrophins (termed “control”), 400 pM BDNF(termed “BDNF-400 pM), 400 pM NT4/5 (termed “NT4/5-400 pM), or 2.5 nMLIF (termed “LIP-2.5 nM).

Example 4: Preparation of Mammalian Expression Vectors and Expression ofAntibody E3 in Mammalian Cells

Three mammalian expression vectors were designed and constructed for usein the expression of antibody E3 in mammalian cells.

Vector db.911.3E is an expression vector comprising the heavy chainvariable region of the E3 antibody and the human IgG2a constant region,and is suitable for transient or stable expression of the heavy chain.db.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); a synthetic intron (nucleotides 619-1507); the DHFRcoding region (nucleotides 707-1267); human growth hormone signalpeptide (nucleotides 1525-1602); antibody 3E heavy chain variable region(nucleotides 1603-1965); human heavy chain IgG2a constant regioncontaining the following mutations: A330P331 to S330S331 (amino acidnumbering with reference to the wildtype IgG2a sequence; see Eur. J.Immunol. (1999) 29:2613-2624); SV40 late polyadenylation signal(nucleotides 2974-3217); SV40 enhancer region (nucleotides 3218-3463);phage f1 region (nucleotides 3551-4006) and beta lactamase (AmpR) codingregion (nucleotides 4443-5300). db.911.3E was deposited at the ATCC onJan. 8, 2003, and was assigned ATCC Accession No. PTA-4895.

Vector Eb.911.3E is an expression vector comprising the light chainvariable region of the E3 antibody and the human kappa chain constantregion, and is suitable for transient expression of the light chain.Eb.911.3E consists of nucleotide sequences corresponding to thefollowing regions: the murine cytomegalovirus promoter region(nucleotides 1-612); human EF-1 intron (nucleotides 619-1142); humangrowth hormone signal peptide (nucleotides 1173-1150); antibody E3 lightchain variable region (nucleotides 1251-1571); human kappa chainconstant region (nucleotides 1572-1892); SV40 late polyadenylationsignal (nucleotides 1910-2153); SV40 enhancer region (nucleotides2154-2399); phage f1 region (nucleotides 2487-2942) and beta lactamase(AmpR) coding region (nucleotides 3379-4236). Eb.911.3E was deposited atthe ATCC on Jan. 8, 2003, and was assigned ATCC Accession No. PTA-4893.

Vector Eb.pur.911.3E is an expression vector comprising the light chainvariable region of the E3 antibody and the human kappa constant region,and is suitable for stable expression of the light chain. Eb.pur.911.3Econsists of nucleotide sequences corresponding to the following regions:the murine cytomegalovirus promoter region (nucleotides 1-612); humanEF-1 intron (nucleotides 619-1758); pac gene (puromycinR) coding region(nucleotides 739-1235); human hsp70 5′UTR region (nucleotides1771-1973); human growth hormone signal peptide (nucleotides 1985-2062);antibody E3 light chain variable region (nucleotides 2063-2383); humankappa chain constant region (nucleotides 2384-2704); SV40 latepolyadenylation signal (nucleotides 2722-2965); SV40 enhancer region(nucleotides 2966-3211); phage f1 region (nucleotides 3299-3654) andbeta lactamase (AmpR) coding region (nucleotides 4191-5048).Eb.pur.911.E3 was deposited at the ATCC on Jan. 8, 2003, and wasassigned ATCC Accession No. PTA-4894.

Transient cell expression was performed as follows: CHO and HEK293Tcells in 150 mm dishes were transiently co-transfected with 25 ug ofeach plasmid (i.e., one plasmid containing the heavy chain and oneplasmid containing the light chain). DNA was mixed with 100 ullipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions. The DNA-lipid complexes were allowed to contact the cellsin DMEM/F12 medium without serum or antibiotics for 5 hours. Followingthis incubation, the media was changed for expression to Opti-MEM(Invitrogen) without any additives for two days. Cell supernatantscontaining antibody were harvested sequentially up to four times withsubsequent media replacement. Supernatants were purified by affinitychromatography using MapSelect Protein A resin (Amersham biosciences17-5199-02). Antibody was bound to the protein A resin in 0.3M glycine,0.6M NaCl buffer at pH 8, then eluted with 0.1 M citrate buffer at pH 3.Fractions containing antibody were immediately neutralized with 1M Trisbuffer at pH 8.0, Antibody fractions were then dialyzed and concentratedin PBS.

Example 5: Anti-NGF Antibody E3 is Effective in Treating Post-SurgicalPain

We used a pain model that mimics post surgical pain to assess theefficacy of treatment with antibody E3. Antibody E3 comprised the humanheavy chain IgG2a constant region containing the following mutations:A330P331 to S330S331 (amino acid numbering with reference to thewildtype IgG2a sequence; see Eur. J. Immunol. (1999) 29:2613-2624); thehuman light chain kappa constant region; and the heavy and light chainvariable regions shown in Tables 1A and 1B.

Animals. Male Sprague Dawley rats weighting between 220-240 grams werepurchased from Harlan (Wisconsin) and acclimated to the animal facilityfor one week prior to surgery.

Surgery. The surgery was based on the procedure described by Brennan, etal. Pain 64:493-501 (1996). Animals were anesthetized with a 2%isoflurane in air mixture that was maintained during surgery via a nosecone. The plantar surface of the right hind paw was prepared with apovidone-iodine pad, and a 1-cm central longitudinal incision was madethrough skin and fascia, starting 0.5 cm from the edge of the heel andextending toward the toes. Measurements were made with a ruler with thefoot held in a flexed position. The plantaris muscle was elevated usingcurved forceps and incised longitudinally. The muscle was incisedthrough its full depth, between the origin and insertion. Bleeding wascontrolled throughout surgery by pressure applied through a gauze pad.The wound was closed with two mattress sutures (5-0 ethilon blackmonofilament). These sutures were knotted 5-6 times, with the first knotloosely tied. The wound site was swabbed with bacitracin solution.Animals were allowed to recover and rest in clean cages for two hours ormore before behavioral testing began.

Evaluating resting pain. A cumulative pain score was used to assess painrelated to weight bearing. Animals were placed on a plastic mesh (grid:8 mm²) in clear plastic cages that were elevated on a platform (h: 18″)allowing inspection of the underside of their paws. After a 20 minuteacclimation period, weight bearing was assessed on a scale of 0 to 2. Ascore of 0 was given if the paw was blanched or pressed against themesh, indicating full weight bearing. A score of 1 was given if the pawwas favored with the skin just touching the mesh, with no blanching orindentation of the skin. A score of 2 was given if the paw was heldcompletely off the mesh. Flinching the paw was considered a 2 if the ratwas still at rest. Each animal was observed for 1 minute every 5 minutesfor 30 minutes. The sum of 6 scores (0-12) obtained during 1/2-hour wasused to assess pain in the incised foot. Frequency of scores of 2 wasalso calculated and used to assess the incidence of severe pain or totalguarding of the paw by the animal. Each animal was tested 24 hoursbefore surgery (baseline), and 2 h, 24 h, 48 h, and 72 hpostoperatively. The results of this experiment are shown in FIG. 1,which depicts the cumulative resting pain score observed in animalstreated with 35 mg/kg of anti-NGF mouse antibody 911. These resultsdemonstrated that treatment with anti-NGF antibody significantly reducedpost-surgical resting pain. Weight bearing was a good correlate of howwilling the animal was to use the limb, and therefore was an effectivemeasure of pain relief.

The E3 antibody was injected intra peritoneal (i.p.) at variousconcentrations of the antibody (0.004, 0.01, 0.02, 0.1, 0.6, and 1 mgper kilogram of animal weight) at 15 hours pre-incision. The negativecontrol group received no antibody but was injected i.p. with a salinesolution. Fentanyl at 0.01 mg/kg was injected i.p. as a positive control30 minutes before testing at 24 hours post-surgery. Each experimentinvolved 8 animals (n=8 per group) for each condition, and the controlgroup had 56 animals. Surgery was performed and a cumulative pain scorewas measured as described above. Resting pain was evaluated twenty-fourhours after the surgery.

As shown in FIG. 7, humanized anti-NGF antibody E3 significantly reducedresting pain (p<0.05) after surgery when administered at 0.02 mg/kg to 1mg/kg dosage. A “*” denotes a significantly significant difference fromcontrol (p<0.05). Treatment with 0.02 mg/kg alleviated pain behavior atleast as effectively as treatment with 0.01 mg/kg fentanyl. This dose offentanyl is 10 times the normal human dose of this potent opioid.

In another experiment, the efficacy of the E3 antibody in reducingpost-surgical pain when administered post-surgically was tested.Antibody E3 (0.5 mg/kg) were injected intravenously (i.v.) two hoursafter surgery. The control group received no antibody but was injectedi.v. with a saline solution. Surgery was performed and resting painexpressed as a cumulative pain score was assessed 24 hours aftersurgery. As shown in FIG. 8, treatment with anti-NGF antibodysignificantly (p<0.05) reduced resting pain at twenty-four hours afterincision when the antibody was administered 2 hours post-incision. Theseresults demonstrated that E3 antibody effectively alleviatedpost-surgical pain when administered after surgery.

Example 6: Assessment of Analgesic Effects of Anti-NGF AntagonistAntibody 911 in a Rat Model of Rheumatoid Arthritis

The analgesic effects of anti-NGF antibody, 911 (see Hongo et al.,Hybridoma 19(3):215-227 (2000)) in complete Freund's adjuvant(CFA)-induced chronic arthritis in rats were investigated using thevocalization test, in comparison with indomethacine used as referencesubstance.

Fifty (50) male Lewis rats (LEWIS LEW/Crl Ico) (Charles River Belgium)weighing 150 g to 220 g at the beginning of the experimental phase wereincluded in this study. All animals were kept for at least 5 days beforethe experiment, and were housed in a temperature (19.5-24.5° C.),relative humidity (45-65%) and 12-h light/dark cycle-controlled roomwith ad libitum access to filtered tap-water and standard pelletedlaboratory chow (U.A.R., France) throughout the study. Animals wereindividually identified on the tail.

On day 0 (D0), arthritis was induced in rats by intradermal injectioninto the tail of 0.05 ml of a Mycobacterium butyricum (Difco, USA)suspension in mineral oil (10 mg/ml). On day 14 (D14), arthritic ratswere included in the study according to their ability to vocalize upongentle flexion of the hindpaw and by their arthritis index, evaluatedusing an inflammation score for each hind and forepaw (see Kuzuna etal., Chem. Pharm. Bull. (Tokyo) 23:1184-1191 (1975); Pearson et al.,Arthritis Rheum. 2:440-459 (1959)). Animals were scored based on thefollowing criteria: Score 0: normal aspect; Score 1: erythema; Score 2:erythema with slight edema; Score 3: strong inflammation withoutankylosis; Score 4: ankylosis. Only animals able to vocalize upon gentleflexion and presenting a score of 2 or 3 were included in the study.

Four groups of 10 rats each were included in the study. For group 1(vehicle), on day 14 (D14), after selection, rats were intravenouslyadministered by vehicle (saline). On day 18 (D18), the nociceptiveintensity was evaluated by gentle flexion of the hindpaw and theintensity of the level of vocalization was recorded for each animal. Forgroup 2 (4 days), on D14, after selection, rats were intravenouslyadministered 911 (10 mg/kg). On day 18 (D18), the nociceptive intensitywas evaluated by gentle flexion of the hindpaw and the intensity of thelevel of vocalization was recorded for each animal. For group 3 (24hours), on day 17 after injection of CFA, rats were intravenouslyadministered 911 (10 mg/kg). The nociceptive intensity was evaluated bygentle flexion of the hindpaw 24 hours later, and the intensity of thelevel of vocalization was recorded for each animal. For group 4(indomethacin), on day 18 (D18), the nociceptive intensity was evaluatedby gentle flexion of the hindpaw one hour after oral administration ofindomethacin (10 mg/kg). The intensity of the level of vocalization wasalso recorded for each animal. The test substances were administered ina blind and random manner by intravenous route under a volume of 5ml/kg, whereas indomethacin was administered by oral route under avolume of 10 ml/kg.

The analgesic effects of anti-NGF antibody 911 are shown in Table 10.The results were expressed for each group as the nociceptive intensityevaluated the intensity of the level of vocalization recorded for eachanimal in mV (mean±SEM), and the percentage of variation of thenociceptive intensity calculated from the mean value of thevehicle-treated group. Statistical significance between the treatedgroups and the vehicle group was determined with a Dunnett's test usingthe residual variance after a one-way analysis of variance (P<0.05).

TABLE 10 Analgesic effects of 911 in complete freund's adjuvant-inducedchronic arthritis in rats Substances (Day of dosing) Vehicle (D14) 911(D14) 911 (D17) Indomethacin (D18) Dose (mg/kg)   10   10   10Nociceptive 971.0 ± 116.2 234.7 ± 34.4 * 247.2 ± 41.8 * 145.8 ± 29.9 *intensity (mV) % variation — −76 −75 −85 Results are expressed as mean ±sem n = 10 rats per group Day 0 (D0): Induction of Chronic arthritis byadministration of CFA Vehicle: saline 911 (10 mg/kg) was intravenouslyadministered at D14 or D17 and pain measurement was performed at D18.Indomethacin (10 mg/kg) was orally given at D18 and pain measurement wasperformed one hour after dosing. Dunnett's test: * indicates asignificant difference in comparison with the vehicle-treated group forP < 0.05

As shown in Table 10, anti-NGF antibody 911 significantly reduced painin a rat model of rheumatoid arthritis 24 hours or 4 days after a singleadministration of the antibody.

Example 7: Pharmacological Effects of Anti-NGF Antagonist Antibody E3and 911 in a Rat Model of Rheumatoid Arthritis

Pharmacological effects (anti-inflammatory and analgesic effects) ofanti-NGF antagonist antibody E3 and 911 were investigated in a model ofcomplete Freund's adjuvant (CFA)-induced chronic arthritis in rats incomparison with indomethacin used as an internal positive controlsubstance. Analgesic effects of E3 and 911 were evaluated by themeasurement of nociceptive response. Anti-inflammatory effects wereevaluated by paw volume, arthritis index (inflammation score), body andhindpaws weight. Paw cytokine levels (IL-6, IL-1β, TNF-α and TGF-β1),circulating TGF-β1 in serum, E3 and 911 plasma concentrations,biological parameters and X-ray radiographies were performed at the endof experiment.

Experimental Protocol 1. Study Design

80 male Lewis rats (LEWIS Lew/Ico) (Charles River Laboratories—Belgium)5-weeks old were included in this study. They were housed in atemperature (19.5-24.5° C.) and relative humidity (45-65%) controlledroom with a 12-h light/dark cycle, with ad libitum access to filteredtap-water and standard pelleted laboratory chow (SAFE, France)throughout the study. Upon receipt at animal facilities, they werehoused 5 per cage and a 10-day acclimatization period were observedbefore any testing. Animals were individually identified on the tail.

Five groups of 10 animals (5-weeks old male Lewis rats—LEWIS Lew/Ico,from Charles River Laboratories—Belgium) each were included in thisstudy: Group 1: non arthritic rats/saline (vehicle), i.v. bolus, n=10;Group 2: arthritic rats/saline (vehicle), i.v. bolus, n=10; Group 3:arthritic rats/Indomethacin 3 mg/kg, p.o daily over 10 days, n=10; Group4: arthritic rats/E3, 1 mg/kg, i.v. bolus, n=10; Group 5: arthriticrats/911, 10 mg/kg, i.v. bolus, n=10. The doses were expressed in termsof free active substance (mg/kg). E3 and 911 were extemporaneouslyprepared in saline from the stock solution to the desired concentration.E3 1 mg/kg: 3.41 mL of the stock solution (0.88 mg/ml) q.s.p. 15 mL ofsaline. 911 10 mg/kg: 12 mL of the stock solution (2.5 mg/ml) q.s.p. 15mL of saline. All diluted solutions (before i.v. injection) weresterilized using a sterile filter unit of 0.20 μm. pH and osmolarityvalues of diluted solutions were measured before each i.v. injection.Before the first i.v., osmolarity (mosm/L) for saline, E3, and 911 were278, 269, and 308 respectively; pH for saline, E3, and 911 were 5.93,6.76, 6.71 respectively. Before the second i.v., osmolarity (mosm/L) forsaline, E3, and 911 were 280, 270, and 309 respectively; pH for saline,E3, and 911 were 5.86, 6.72, and 6.59 respectively.

E3 or 911 or saline were administered by i.v. bolus injection on Day 14and Day 19 after arthritis induction in a coded and random order with avolume of 5 mL/kg. The non arthritic group was given by i.v. bolusinjection of saline on Day 14 and Day 19 with a volume of 5 mL/kg.Indomethacin was extemporaneously prepared in 1% methylcellulose.Indomethacin was administered by oral route (p.o.) once daily over 10days from Day 14 to Day 23 after arthritis induction in a coded andrandom order with a volume of 10 mL/kg.

2. Induction of Arthritis

On Day 0 (D 0), arthritis was induced in 70 rats by intradermalinjection into the tail of 0.05 ml of a Mycobacterium butyricumsuspension. A group of 10 rats did not receive any intradermal injection(non arthritic rats). On Day 14 (D 14), the arthritic rats were includedin the study using the following criteria: all included rats displayedan increase of mean paw volume (mean of the left and right paw volume)of at least 0.30 ml compared to the mean paw volume (mean of the leftand right paw volume) in the non arthritic group (paw volume measurementas described below); all included rats displayed a vocalization upongentle flexion (nociceptive response measurement as described below);and all included rats displayed a score of arthritis index of 2-3 oneach hindpaw (arthritis index measurement as described below) (theanimals with a score of 0, 1 or 4 were discarded).

3. Body Weight

The animals were weighed once daily from Day 0 to Day 24 (except duringthe week-end days before the treatment: D 1, D 2, D 8, D 9, D10). Allmeasurements were performed between 9:00 and 12:00 am except at D 14(7:30-9:00 am) and D 24 (7:30-8:00 am).

3. Paw Volume Measurement

The right and left hindpaw volume of each rat (arthritic and nonarthritic rats) was measured using a plethysmometer. The measurementswere performed at the following times (after induction of arthritis):Day 14 (before i.v. bolus or p.o. administration); and Day 24 (5 daysafter the last i.v. bolus injection or 24 h after the last p.o.administration). All measurements were performed between 9:00 and 12:00am. All the data were collected and stored by the WinDas software.

4. Arthritis Index

Arthritis index was evaluated using an inflammation score for each hindand forepaw (arthritic rats): Score 0: normal aspect; Score 1: erythema;Score 2: erythema with slight edema; Score 3: strong inflammationwithout ankylosis; Score 4: ankylosis. This evaluation was performed atthe following times (after induction of arthritis): Day 14 (before i.v.bolus or p.o. administration); and Day 24 (5 days after the last i.v.bolus injection or 24 h after the last p.o. administration). Allmeasurements were performed between 2:00 and 3:00 pm (D 14), 8:00 and9:00 am (D 24). All the data were collected and stored by the WinDassoftware.

5. Measurement of Nociceptive Response (Vocalization Test)

The nociceptive response was evaluated by gentle flexion of the rightand left hindpaw repeatedly 2 times at intervals of 4 to 5 sec with afinger of the operator (arthritic rats). The intensity of the level ofvocalization was recorded for each animal for each hindpaw (2 times: onright hindpaw: s1 and s3; 2 times: on left hindpaw: s2 and s4). Thisevaluation was performed at the following times (after induction ofarthritis): Day 14 (before i.v. bolus or p.o. administration); Day 18(before the second i.v. bolus injection or 1 hr after p.o.administration); and Day 24 (5 days after the last i.v. bolus injectionor 24 h after the last p.o. administration). All measurements wereperformed between 9:00 and 12:00 am except at D 14 (7:30-9:00 am) and D24 (7:30-9:00 am).

6. Blood Collection for Measurement of E3 or 911 Concentration andCirculating TGF-β1 and Hematological Parameters

On Day 24 (after paw volume and arthritis index measurements and testvocalization), under general anaesthesia using isoflurane (in a mixtureof oxygen and nitrous oxide), the blood samples (about 800-1000 μl) wascollected by capillary action with a micropipette from retroorbitalsinus.

Measurement of E3 or 911 concentration (groups 2, 4 and 5): A part ofblood sample was collected in tubes containing Li-Heparin (maintained onice) and centrifuged at 2500-3000 g for 10 min. Plasma samples (at least100 μL) were obtained, frozen in liquid nitrogen, stored at −80° C. Onesample was slightly hemolyzed (vehicle-treated arthritic rat #36).

Measurement of circulating TGF-β1 (groups 1-2-3-4-5): A part of bloodsample was collected in micro tubes for serum preparation at ambienttemperature. Following sample collection, blood was mixed and allowed toclot for 30 minutes prior to the centrifugation. The tubes werecentrifuged at about 6000 g for 3 minutes. Each serum sample (at least100 μL, except for rat #52 and #53) was aliquoted and stored at −20° C.until sample activation for TGF-β1 analysis. These aliquots (50 vials)were kept for a period of 6 months starting from the end of the study.Some samples were slightly hemolyzed (vehicle-treated non arthritic rat:#2, #5, #9, #10; vehicle treated arthritic rat: #53, #63; E3-treatedarthritic rat #31, #51; 911-treated arthritic rat: #52, 62, #64). TGF-β1levels were measured using human TGF-β1 ELISA kit (ref DB100, Batch212258 and 213610, R&D Systems—France).

Blood collection for hematological parameters (groups 1-2-3-4-5: 50vials): A part of blood sample was collected in tubes containing K3-EDTA(at least 100 μL). The determination of parameters were performed on theday of the collection and the samples were not stored. The hematologicalparameters including red blood cells, white blood cells, platelets,hemoglobin, hematocrit were measured with a hematology cell counter (D24). Some hematological parameters were not measured due to the clottedsamples (vehicle-treated non arthritic rat: #10; E3-treated arthriticrats: #59, #67; 911-treated arthritic rats: #16).

7. Paw Cytokines Levels

On Day 24 (5 days after the last i.v. bolus injection or 24 hours afterthe last p.o. administration) (after X-rays radiographies), each animalhindpaw (arthritic and non arthritic rats) was weighed and was collectedin a labelled polyethylene vial. Tissue samples were frozen in liquidnitrogen and stored at −80° C.

Preparation of joint homogenates: Frozen hind paws were pulverized usinga Bio-Pulverizer. The powdered hind paws were then placed into a 50 mlconical centrifuge tube containing 3 ml PBS supplemented with 50 μl ofanti-protease cocktail and homogenized on ice using Ultra-Turraxhomogenizer (50% of the maximal speed). Homogenates were thencentrifuged at 2000×g for 15 minutes at 4° C. and supernatants werefiltered through 0.2 μm Sartorius filters, aliquoted and stored at −80°C. until use.

Cytokine levels measurement: Cytokine levels of TNF-α (Rat TNF-α ELISAkit, ref RTA00, Batch 213718, R&D Systems, France), IL-1β Rat IL-1βELISA kit, ref. RLB00, Batch 212435, R&D Systems, France), IL-6 Rat IL-6ELISA kit, ref. R6000, Batch 211773, 214008 and 214362, R&D Systems,France), and TGF-β1 Human TGF-β1 ELISA kit, ref. DB100, Batch 212258 and213610, R&D Systems, France) were determined in duplicate, according tothe manufacturer's procedure. Aliquots of hind paw homogenates werestored at −80° C.

8. X-Ray Analysis

On Day 24, after blood collecting the animals were sacrificed and X-rayradiographies (hindpaws) were obtained for assessment of joint lesions.X-ray analysis was focused on articular erosions, articular space,periosteum abnormalities on both hindpaws. All the radiographies wereanalyzed by looking at seven different items: the soft tissue damage,deformity, demineralization, joint space, erosions, osteogenesis andperiostal reaction. For each animal, the first six items were analyzedindependently by looking at the worse hind foot. The periostal reactionwas analyzed by looking at the tail. For each item, the score goes from0 (normal) to 4 (maximal damage). Therefore the total score goes from 0to 28. The radiographic interpretation was done by the same readerwithout knowing anything about the animals (treated or not treated).

9. Observations

One animal (#65) died at D 23 after indomethacin administration (beforethe administration at D 23) due to an unknown cause.

10. Analysis and Expression of Results

All results were reported as Mean S.E.M. of 10 rats in each group ateach time point. Paw volume was expressed in ml calculated from the meanvalue of the right and left paw volume. Arthritis index was calculatedfrom the sum of the score obtained for each of the 4 paws. Thenociceptive response was evaluated by the intensity of the level ofvocalization recorded for each animal (mean of 4 values: 2 times/paw) inmV. The percentage inhibition of the nociceptive response was calculatedfrom the mean value of the vehicle-treated arthritic group [(mean valueof vehicle-treated arthritic group−mean value of treated arthriticgroup/mean value of vehicle-treated arthritic group)*100]. Body weightwas expressed in grams. Hindpaws (left and right) weight was expressedin grams. Cytokine levels (IL-6, IL-1β, TNF-α and TGF-β1) of each hindpaw was expressed in pg/ml. Circulating levels of TGF-β1 was expressedin pg/ml. Radiological index for each parameter (demineralization,erosions, periostal reaction, soft tissue damage, space joint,osteogenesis deformity) and total radiological index (total score) werecalculated from the sum of the scores obtained for each parameter. Theinter-group significances of the deviations between the values ofvehicle-treated group (arthritic rats) and vehicle-treated group (nonarthritic rats) were assessed by the Student t test or Mann-Whitney RankSum Test when equal variance or normality test failed. The inter-groupsignificances of the deviations between the values of vehicle-treatedgroup (arthritic rats) and E3- and 911- and Indomethacin-treated groupswere assessed by the 1-way analysis of variance ANOVA followed by thenon-paired Dunnett t test. A probability of P<0.05 was considered assignificant. All statistical analysis was performed by the Sigmastat™software.

Results 1. Nociceptive Response (Vocalization Test)

As shown in Table 11 and FIG. 18, on D 14, the nociceptive response was4147±331, 4386±235, 4644±367 and 4468±143 in vehicle-, indomethacin-,E3-, and 911-treated arthritic groups, respectively. Indomethacinstrongly and significantly decreased the nociceptive response after 3mg/kg/day p.o. (for 10 days) by about −3768 mV (% inhibition: 71%) and−4353 mV (% inhibition: 74%) at D 18 and D 24, respectively compared tothe vehicle-treated arthritic group (D 18: 1511±398 vs 5279±326 mV; D24: 1552±508 vs 5905±345 mV). E3 (1 mg/kg i.v. at D 14 and D 19)strongly and significantly decreased the nociceptive response by about−4167 mV (% inhibition: 79%) and −5905 mV (% inhibition: 100%) at D 18and D 24, respectively compared to the vehicle-treated arthritic group(D 18: 1112±401 vs 5279±326 mV; D 24: 0±0 vs 5905±345 mV). 911 (10 mg/kgi.v. 2 days at D 14 and D 19) strongly and significantly decreased thenociceptive response by about −3932 (% inhibition: 74%) and −5358 mV (%inhibition: 91%) at D 18 and D 24, respectively compared to thevehicle-treated arthritic group (D 18: 1347±492 vs 5279±326 mV; D 24:547±307 vs 5905±345 mV).

TABLE 11 Effects of E3 and 911 after i.v. injection (2 days: D 14-D 19)on nociceptive response in rheumatoid arthritis in rats Day D14 D18 D24Arthritic Rats vehicle i.v. 4147 ± 331 5279 ± 326 5905 ± 345 E3 4644 ±367 1112 ± 401 *   0 ± 0 * 1 mg/kg i.v. % inhibition 0 79 100 911 4468 ±143 1347 ± 492 *  547 ± 307 * 10 mg/kg i.v. % inhibition 0 74  91Indomethacin 4386 ± 235 1511 ± 398 * 1552 ± 508 3 mg/kg p.o. (over 10days) % inhibition 0 71  74 Values are expressed in mV as Mean ± S.E.M.n = 10 animals per group except at D 24 for Indomethacin (n = 9) Dunnettt test: * P ≤ 0.05 vs vehicle-treated arthritic rats

2. Body Weight

As shown in Table 12 and FIG. 19, a marked decrease in the body weightgain was observed in arthritic rats in comparison to non arthritic ratsfrom D 0 to D 14 due to arthritis establishment. At D 14 (selection day)the arthritic rats displayed a significant decrease in weight comparedto the non arthritic rats (289±2 vs 217±4 g) (Student t test P<0.05).However, no significant difference in weight (D 14) was detected in allarthritic groups (Dunnett t test P>0.05). The body weight moderately andsignificantly increased in Indomethacin-treated group (3 mg/kg/day for10 days) from D 17 to D 24 with a maximum of about 43 g at D 24 comparedto the vehicle-treated arthritic group (261±5 vs 218±3 g). After E3treatment (1 mg/kg i.v. at D 14 and D 19), the body weight moderatelyand significantly increased from D 17 to D 24 with a maximum of about 46g at D 24 compared to the vehicle-treated arthritic group (264±5 g vs218±3 g). After 911 treatment (10 mg/kg i.v. at D 14 and D 19), the bodyweight moderately and significantly increased from D 18 to D 24 with amaximum of about 47 g at D 24 compared to the vehicle-treated arthritic(265±7 vs 218±3 g).

TABLE 12 Effects of E3 and 911 after i.v. injection (2 days: D 14-D 19)on body weight in rheumatoid arthritis in rats Day D0 D3 D4 D5 D6 D7 D11D12 D13 D14 Non Arthritic vehicle i.v. 197 ± 215 ± 222 ± 232 ± 236 ± 244± 272 ± 277 ± 282 ± 289 ± Rats 2 2 2 2 2 2 2 2 2 2 Arthritic vehiclei.v. 199 ± 214 ± 221 ± 230 ± 236 ± 241 ± 229 ± 223 ± 218 ± 217 ± Rats 22 2 2 2 3 6 5 5 4 E3 206 ± 222 ± 230 ± 241 ± 243 ± 249 ± 242 ± 237 ± 230± 225 ± 1 mg/kg i.v. 4 3 3 3 3 3 6 6 5 5 911 201 ± 211 ± 218 ± 227 ± 231± 239 ± 234 ± 228 ± 221 ± 218 ± 10 mg/kg i.v. 2 5 5 5 5 5 8 7 7 6Indomethacin 202 ± 217 ± 225 ± 235 ± 239 ± 246 ± 242 ± 235 ± 227 ± 224 ±3 mg/kg p.o. 3 4 4 4 4 4 7 7 6 5 over 10 days Day D15 D16 D17 D18 D19D20 D21 D22 D23 D24 Non Arthritic vehicle i.v. 285 ± 291 ± 297 ± 302 ±307 ± 308 ± 312 ± 316 ± 321 ± 326 ± Rats 2 2 2 3 3 3 3 3 3 3 Arthriticvehicle i.v. 213 ± 212 ± 211 ± 210 ± 208 ± 210 ± 212 ± 214 ± 216 ± 218 ±Rats 4 4 3 3 3 3 3 3 3 3 E3 223 ± 224 ± 227 ± 232 ± 235 ± 238 ± 245 ±250 ± 257 ± 264 ± 1 mg/kg i.v. 5 5 4 4 4 4 4 5 5 5 * * * * * * * * 911217 ± 221 ± 226 ± 229 ± 233 ± 239 ± 246 ± 253 ± 258 ± 265 ± 10 mg/kgi.v. 5 5 5 5 6 6 6 6 6 7 * * * * * * * Indomethacin 230 ± 230 ± 231 ±234 ± 236 ± 241 ± 246 ± 248 ± 253 ± 261 ± 3 mg/kg p.o. 4 5 4 4 4 4 4 5 55 over 10 days * * * * * * * * Values are expressed in grams as MeanS.E.M. n = 10 animals per group except at D 23 and D 24 (n = 9) forIndomethacin Dunnett t test: * P ≤ 0.05 vs vehicle-treated arthriticrats

3. Paw Volume

On D 14, a randomization was performed in order to obtain homogenousgroups in terms of paw volume. As shown in Table 13, on D 14, thehindpaw volume (mean of the right and left paw volume) was significantlygreater in arthritic group than that in non arthritic group (2.10±0.05vs 1.44±0.02 mL (Student t test P<0.05)). Indomethacin (3 mg/kg/day p.o.for 10 days) significantly decreased the paw volume by about −0.75 mL (D24) compared to the vehicle-treated arthritic group (1.59±0.03 mL vs2.34±0.08 mL). E3 (1 mg/kg i.v. on D 14 and D 19) slightly andsignificantly increased the paw volume by about 0.37 mL compared to thevehicle-treated arthritic group (2.71±0.09 mL vs 2.34±0.08 mL). 911 (10mg/kg i.v. on D 14 and D 19) slightly and significantly increased thepaw volume by about 0.36 mL compared to the vehicle-treated arthriticgroup (2.70±0.11 mL vs 2.34±0.08 mL).

TABLE 13 Effects of E3 and 911 after i.v. injection (2 days: D 14-D 19)on paw volume in rheumatoid arthritis in rats Day D14 D24 Non vehiclei.v. 1.44 ± 0.02 1.47 ± 0.02 Arthritic Rats Arthritic vehicle i.v. 2.10± 0.05 2.34 ± 0.08 Rats E3 2.06 ± 0.03 2.71 ± 0.09 * 1 mg/kg i.v. 9112.02 ± 0.07 2.70 ± 0.11 * 10 mg/kg i.v. Indomethacin 2.08 ± 0.06 1.59 ±0.03 * 3 mg/kg p.o. over 10 days Values are expressed in mL as Mean ±S.E.M. n = 10 animals per group except at D 24 for Indomethacin (n = 9)Dunnett t test: * P ≤ 0.05 vs vehicle-treated arthritic rats

4. Arthritis Index

As shown in Table 14, on D 14, the arthritis index was 10.1±0.8,8.7±0.6, 10.2±0.4 and 9.4±0.7 and in vehicle-indomethacin-, E3-, and911-treated arthritic groups, respectively. Indomethacin strongly andsignificantly decreased the arthritis index after 3 mg/kg/day p.o. (for10 days) by a maximum of about −8.0 compared to the vehicle-treatedarthritic group (2.7±0.7 vs 10.7±0.6). E3 (1 mg/kg i.v. on D 14 and D19) did not affect the arthritis index compared to the vehicle-treatedarthritic group (11.4±0.4 vs 10.7±0.6). 911 (10 mg/kg i.v. on D 14 and D19) did not affect the arthritis index compared to the vehicle-treatedarthritic group (10.9±0.7 vs 10.7±0.6).

TABLE 14 Effects of E3 and 911 after i.v. injection (2 days: D 14-D 19)on arthritis index in rheumatoid arthritis in rats Day D14 D24 Arthriticvehicle i.v. 10.1 ± 0.8 10.7 ± 0.6 Rats E3 10.2 ± 0.4 11.4 ± 0.4 1 mg/kgi.v. 911  9.4 ± 0.7 10.9 ± 0.7 10 mg/kg i.v. Indomethacin  8.7 ± 0.6 2.7 ± 0.7 * 3 mg/kg p.o. over 10 days Values are expressed as Mean ±S.E.M. (score) n = 10 animals per group except for Indomethacin (n = 9)Dunnett t test: * P ≤ 0.05 vs vehicle-treated arthritic rats

5. Paw Cytokines Levels

As shown in Table 15, on D 24, the left and right paws cytokine levelswere increased in arthritic vehicle-treated group by a maximum of about3.5 (IL-1β), 4 (TNF-α) and 1.8 (TGF-β1) fold compared to thenon-arthritic vehicle-treated group. No significant difference wasobserved for IL-6 levels, in right and left paw, between the two groups.The cytokines levels of arthritic group were similar in left and rightpaw: 259.7±38.5 vs 219.2 32.4, 4802.8±365.5 vs 4007.1±380.4, 17.8±1.6 vs18.6±1.9 and 9735.0±1219.8 vs 9161.4±846.1 μg/ml for IL-6, IL-1β, TNF-αand TGF-β1 respectively. Indomethacin slightly, but significantly,decreased the TGF-β1 level in right paw after 3 mg/kg/day p.o. (for 10days) by about 1.3 times, compared to the vehicle-treated arthriticgroup (7057.4±335.6 vs 9161.4±846.1), whereas it did not modify IL-6,TNF-α or IL-1β levels. A similar but not significant effect was observedin the left paw. E3 (1 mg/kg i.v. on D 14 and D 19) did not affect theIL-6, IL-1β, TNF-α or TGF-β1 levels, in both paws, compared to thevehicle-treated arthritic group. 911 (10 mg/kg i.v. on D 14 and D 19)increased the IL-1β level in right paw compared to the vehicle-treatedarthritic group (6215.3±666.7 vs 4007.1±380.4). It had no effect onothers cytokine levels in both paws.

Table 15. Effect of E3 and 911 after i.v. Injection (2 Days on D 14 andD 19) on Paw Cytokines Levels in Rheumatoid Arthritic Rats

Left paw cytokines levels Arthritic Rats Non-arthritic Rats E3 911Indomethacin vehicle i.v. vehicle i.v. 1 mg/kg i.v. 10 mg/kg i.v. 3mg/kg p.o. IL-6  298.6 ± 35.6  259.7 ± 38.5  234.4 ± 35.2  262.5 ± 42.5 249.7 ± 60.4 IL-1β 1383.0 ± 57.9 4802.8 ± 365.5 5060.0 ± 473.5  5500.8± 625.3 4029.1 ± 449.9 TNF-α   4.3 ± 2.9  17.8 ± 1.6  23.6 ± 2.5   29.9± 4.8  29.9 ± 3.6 TGF-β1 5264.7 ± 209.2 9735.0 ± 1219.8 9796.7 ± 491.211053.5 ± 713.3 7708.2 ± 293.9 IL-6  286.4 ± 76.1  219.2 ± 32.4  214.6 ±47.2  284.9 ± 38.9  295.9 ± 47.8 IL-1β 1342.1 ± 86.1 4007.1 ± 380.44853.5 ± 605.0  6215.3 ± 666.7 * 3884.4 ± 534.4 TNF-α  15.7 ± 4.8  18.6± 1.9  21.5 ± 2.5   33.4 ± 5.7  30.6 ± 5.7 TGF-β1 5024.8 ± 148.4 9161.4± 846.1 9362.7 ± 423.4 10861.2 ± 604.6 7057.4 ± 335.6 * Values areexpressed in pg/ml, as Mean ± S.E.M. n = 10 animals per group except forNon-arthritic/vehicle (Right paw), Arthritic/vehicle (Left paw) andIndomethacin (n = 9) Dunnett t test: * P ≤ 0.05 vs vehicle-treatedarthritic rats

6. Measurement of Circulating TGF-β1

As shown in Table 16, on D 24, the serum TGF-β1 level was increased inarthritic vehicle-treated group compared to the non arthriticvehicle-treated group (81715.7±1984.1 vs 60269.9±2142.8). Indomethacinsignificantly decreased the serum TGF-β1 level after 3 mg/kg/day p.o.(for 10 days) by about 1.5 times, compared to the vehicle-treatedarthritic group (57222.2±3194.1 vs 81715.7±1984.1). E3 (1 mg/kg i.v. onD 14 and D 19) and 911 (10 mg/kg i.v. on D 14 and D 19) significantlydecreased the serum TGF-β1 level so that the cytokine level in E3- and911-treated groups were comparable with those observed invehicle-treated non arthritic group (69408.8±3926.7 and 67214.5±3649.4respectively, vs 60269.9±2142.8).

TABLE 16 Effect of E3 and 911 after i.v. injection (2 days on D 14 and D19) on serum TGF-β1 levels in rheumatoid arthritic rats Arthritic RatsNon-arthritic Rats E3 911 Indomethacin vehicle i.v. vehicle i.v. 1 mg/kgi.v. 10 mg/kg i.v. 3 mg/kg p.o. TGF-β1 60269.9 ± 2142.8 81715.7 ± 1984.169408.8 ± 3926.7 * 67214.5 ± 3649.4 * 57222.2 ± 3194.1 * Values areexpressed in pg/ml, as Mean ± S.E.M. n = 10 animals per group except forNon-arthritic/vehicle (Right paw), Arthritic/vehicle (Left paw) andIndomethacin (n = 9) Dunnett t test: * P ≤ 0.05 vs vehicle-treatedarthritic rats

7. Hematological Parameters

As shown in Table 17, the hematological parameters such as white bloodcells and platelets were greater in vehicle-treated arthritic rats incomparison to vehicle-treated non arthritic rats (Student t testP<0.05), whereas the red blood cells, hemoglobin and hematocrit (Studentt test P>0.05) were unchanged. Indomethacin did not affect the bloodparameters after 3 mg/kg/day p.o. (for 10 days) compared to thevehicle-treated arthritic group. E3 (1 mg/kg i.v. on D 14 and D 19) didnot affect the blood parameters compared to the vehicle-treatedarthritic group. 911 (10 mg/kg i.v. on D 14 and D 19) did not affect theblood parameters compared to the vehicle-treated arthritic group.

TABLE 17 Effects of E3 and 911 after i.v. injection (2 days on D 14 andD 19) on blood parameters in rheumatoid arthritis in rats (Measurementat D 24) White blood Red blood cells cells Hemoglobin HematocritPlatelets Day 10³/mm³ 10⁶/mm³ g/dl % 10³/mm³ Non Arthritic vehicle i.v.8.7 ± 0.9 7.98 ± 0.31 15.1 ± 0.7 42.6 ± 1.6 322 ± 89  Rats n = 9  n = 9 n = 9  n = 9  n = 9  Arthritic Rats vehicle i.v. 19.0 ± 0.9  7.54 ± 0.3113.2 ±0.7 37.4 ± 1.6 10.43 ± 89   n = 10 n = 10 n = 10 n = 10  n = 10 E319.1 ± 1.2  7.74 ± 0.17 12.9 ± 0.3 38.5 ± 1.0 827 ± 77  1 mg/kg i.v. n =7  n = 8  n = 8  n = 8  n = 8  911 22.6 ± 2.9  7.30 ± 0.40 12.1 ± 0.736.5 ± 2.1 799 ± 121 10 mg/kg i.v. n = 8  n = 9  n = 9  n = 9  n = 9 Indomethacin 21.7 ± 2.5  6.93 ± 0.31 11.8 ± 0.6 35.0 ± 1.5 705 ± 111 3mg/kg p.o. n = 9  n = 9  n = 9  n = 9  n = 9  over 10 days Values areexpressed as Mean ± S.E.M. Anova: P > 0.05 vs vehicle-treated arthriticrats

7. Hindpaw Weight

As shown in Table 18, the left and right hindpaw weight was greater invehicle-treated arthritic rats than in vehicle-treated non arthriticrats (3.43±0.11 vs 1.98±0.01 and 3.32±0.12 vs 1.99±0.02 g, respectively)(Student t test or Mann-Withney P<0.05). Indomethacin significantlydecreased the hindpaws weight after 3 mg/kg/day p.o. (for 10 days)compared to the vehicle-treated arthritic group (left hindpaw: 2.23±0.04vs 3.43±0.11 g; right hindpaw: 2.20±0.05 vs 3.32±0.12 g). E3 (1 mg/kgi.v. on D 14 and D 19) only significantly increased the left hindpawweight compared to the vehicle-treated arthritic group (left hindpaw:3.86±0.14 vs 3.43±0.11 g; right hindpaw: 3.72±0.13 vs 3.32±0.12 g). 911(10 mg/kg i.v. on D 14 and D 19) only significantly increased the righthindpaw weight compared to the vehicle-treated arthritic group (lefthindpaw: 3.73±0.12 vs 3.43±0.11 g; right hindpaw: 3.83±0.15 vs 3.32±0.12g).

TABLE 18 Effects of E3 and 911 after i.v. injection (2 days on D 14 andD 19) on hindpaws weight in rheumatoid arthritis in rats (Measurement atD 24) Left paw Right paw Non vehicle i.v 1.98 ± 0.01 1.99 ± 0.02Arthritic Rats Arthritic Rats vehicle i.v. 3.43 ± 0.11 3.32 ± 0.12 E33.86 ± 0.14 * 3.72 ± 0.13 1 mg/kg i.v. 911 3.73 ± 0.12 3.83 ± 0.15 * 10mg/kg i.v. Indomethacin 2.23 ± 0.04 * 2.20 ± 0.05 * 3 mg/kg p.o. over 10days Values are expressed in grams as Mean ± S.E.M. n = 10 animals pergroup except for Indomethacin (n = 9) Dunnett t test: * P ≤ 0.05 vsvehicle-treated arthritic rats

8. X-Ray Analysis

As shown in Table 19, a total score of 0.0±0.0 was observed in thevehicle-treated non arthritic rats. The vehicle-treated arthritic ratshave a total score of 15.1±1.3 with high scores for demineralization(2.4±0.3), erosions (2.7±0.3), soft tissue damage (3.1±0.2) and spacejoint (3.3±0.2), a moderate score for periostal reaction (1.0±0.3),osteogenesis (0.8±0.2) and deformity (1.8±0.2). Indomethacin (3mg/kg/day p.o. for 10 days) strongly and significantly decreased thetotal score by about 10.7 in comparison to vehicle-treated arthriticrats (4.4±0.9 vs 15.1±1.3). E3 (1 mg/kg i.v. on D 14 and D 19) did notaffect the total score compared to the vehicle-treated arthritic group(14.2±1.3 vs 15.1±1.3). 911 (10 mg/kg i.v. on D 14 and D 19) did notaffect the total score compared to the vehicle-treated arthritic group(15.4±1.0 vs 15.1±1.3).

TABLE 19 Effects of E3 and 911 after i.v. injection (2 days on D 14 andD 19) on X-ray parameters in reheumatoid arthritis in rats Soft Deminer-Periostal tissue Space osteo TOTAL Day alization Erosions reactiondamage joint genesis Deformity score Non Arthritic vehicle i.v. 0.0 ±0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0  0.0 ±0.0 Rats Arthritic Rats vehicle i.v. 2.4 ± 0.3 2.7 ± 0.3 1.0 ± 0.3 3.1 ±0.2 3.3 ± 0.2 0.8 ± 0.2 1.8 ± 0.2 15.1 ± 1.3 E3 2.0 ± 0.2 2.4 ± 0.3 0.8± 0.2 3.3 ± 0.3 2.7 ± 0.2 1.2 ± 0.2 1.8 ± 0.2 14.2 ± 1.3 1 mg/kg i.v.911 2.3 ± 0.3 2.5 ± 0.2 1.0 ± 0.3 3.4 ± 0.2 3.3 ± 0.2 0.9 ± 0.2 2.0 ±0.2 15.4 ± 1.0 10 mg/kg i.v. Indomethacin 0.3 ± 0.2 0.9 ± 0.2 0.7 ± 0.31.0 ± 0.2 1.0 ± 0.2 0.1 ± 0.1 0.4 ± 0.2  4.4 ± 0.9 3 mg/kgp.o. * * * * * * Values are expressed as Mean ± S.E.M (score). n+3210animals per group except for Indomethacin (n = 9) Dunnett t test: * P

Conclusion

Under experimental conditions described above, E3 (1 mg/kg i.v. 2 days:D 14-D 19) and 911 (10 mg/kg i.v. 2 days: D 14-D 19) showed stronganalgesic effects, but did not show significant anti-inflammatoryeffects in this arthritis model.

Example 8 Effects of Different Doses of Anti-NGF Antibody E3 in a RatModel of Rheumatoid Arthritis

The ability of E3 to produce reduction in pain in arthritic rats wasfurther investigated by examining the dose response relationship betweenE3 administration and pain reduction. Rats were treated with adjuvant toinduce arthritis as described above. Ten rats not injected with adjuvantwere used as non-arthritic controls. Fourteen days after adjuvantinjection, animals were qualified into the study based on the criteriastated above, randomized into eight groups of ten rats and tested forthe intensity of their vocalization response. They were then dosed onday 14 with saline, or 0.003 mg/kg, 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg,0.3 mg/kg, 1 mg/kg or 5 mg/kg of E3 antibody as described above. Animalswere tested for their vocalization response on days 16, 18, 20, and 24.Animals were redosed with saline or the same dose of E3 on day 18 afterthe vocalization testing. Animals were also weighed each day, startingat day 14. Thus, animals were dosed twice with a given dose of antibodyor saline on days 14 and 18, and assessed for pain five times, on days14, 16, 18, 20 and 24. Data are shown in Tables 20-22 and in FIGS.20-22.

TABLE 20 Effects of different doses of E3 on nociceptive response(vocalization intensity) in rheumatoid arthritic rats. Vocalizationintensity values are expressed in mV as mean ± s.e.m. 0.003 0.01 0.030.1 0.3 1.0 5.0 vehicle mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg day 14mean 1129.25 981.75 1007.28 963.18 1159.30 1191.58 1067.00 896.25 s.e.m143.06 71.00 66.50 62.12 132.76 123.44 69.73 57.53 day 16 mean 1042.85825.60 576.88 448.43 283.71 151.85 98.62 79.18 s.e.m 130.51 57.94 49.7181.01 60.00 26.08 29.17 27.30 day 18 mean 968.10 427.43 334.45 292.52262.96 194.19 174.13 200.42 s.e.m 117.85 48.55 35.10 52.36 62.32 53.5688.61 120.15 day 20 mean 942.18 448.00 313.13 209.48 79.74 66.27 71.2363.57 s.e.m 100.69 33.73 61.98 24.43 33.18 31.34 42.37 23.47 day 24 mean913.68 724.50 596.38 513.60 432.45 176.32 19.21 12.35 s.e.m 131.29115.90 44.76 63.67 70.38 66.61 10.14 12.35

The effect of treating animals with various doses of anti-NGF antibodyE3 on pain induced vocalization (data shown in Table 20) wasstatistically analyzed by using two-way ANOVA to compare the resultsobtained pairwise between arthritic animals treated with vehicle withthose treated with a given dose of antibody E3. There was a highlysignificant effect at all levels of E3 tested (p<0.0001). Even at thelowest dose tested (0.003 mg/kg of E3), the difference in vocalizationwas significant (p<0.0001).

As shown in Table 20 and FIG. 20, in agreement with the aboveexperiments, treatment with antibody E3 at 1 mg/kg showed a rapid androbust relief of pain. Within two days (the earliest time point tested)the vocalization intensity fell by 90%. Treatment with lowerconcentrations of E3 also provided robust pain relief, although at lowerdoses the pain relief took somewhat longer to manifest. It is likelythat the apparent decrease in efficacy on day 24 of all but the highestdoses tested is due to a decrease in the actual level of plasma E3 levelsecondary to an immune response by the subject rats. It is apparent thatdoses as low as 0.003 mg/kg provide at least partial pain relief in thismodel.

TABLE 21 Effects of different doses of E3 on body weight in rheumatoidarthritic rats (normalized to day 14). Non-Arthritic vehicle 0.003 mg/kg0.01 mg/kg 0.03 mg/kg Day Mean S.E.M Mean S.E.M Mean S.E.M Mean S.E.MMean S.E.M 14 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00 100.000.00 15 99.53 0.30 99.14 0.37 99.20 0.48 99.18 0.43 100.34 0.36 16102.52 0.45 99.57 0.60 99.58 0.79 99.33 0.72 100.89 0.57 17 103.31 0.4199.50 0.64 100.46 0.77 99.69 0.73 101.80 0.82 18 106.11 0.72 100.26 0.93100.90 1.19 100.69 0.72 102.70 0.92 20 109.62 0.85 101.46 1.22 102.261.58 102.70 1.07 104.51 0.75 21 110.52 0.93 102.73 1.49 103.16 1.87102.63 1.18 105.08 0.98 23 114.28 1.19 104.54 1.92 106.09 1.67 104.411.33 106.14 1.06 24 115.44 1.15 105.12 1.92 106.16 1.90 104.23 1.46106.23 1.26 0.1 mg/kg 0.3 mg/kg 1.0 mg/kg 5.0 mg/kg Day Mean S.E.M MeanS.E.M Mean S.E.M Mean S.E.M 14 100.00 0.00 100.00 0.00 100.00 0.00100.00 0.00 15 99.83 0.59 101.05 0.38 100.53 0.37 101.61 0.41 16 101.070.82 102.88 0.50 102.95 0.56 104.09 0.60 17 101.89 1.12 104.76 0.70105.74 0.76 106.85 0.79 18 103.69 1.47 107.11 0.78 108.46 0.82 109.531.00 20 107.36 1.78 111.26 0.77 113.57 0.83 115.32 1.11 21 108.50 2.01113.31 0.87 116.71 0.92 119.11 1.21 23 109.25 2.15 115.59 1.38 123.351.13 126.36 1.94 24 108.77 2.08 115.58 1.43 124.41 1.00 127.25 1.79

TABLE 22 Effects of different doses of E3 on body weight in rheumatoidarthritic rats (normalized to day 0). Non-Arthritic vehicle 0.003 mg/kg0.01 mg/kg 0.03 mg/kg Day Mean S.E.M Mean S.E.M Mean S.E.M Mean S.E.MMean S.E.M 0 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.00 100.00 0.001 100.45 0.19 98.34 0.48 98.37 0.35 98.86 0.33 98.67 0.34 2 105.94 0.33101.75 0.71 102.47 0.59 102.61 0.40 102.05 0.53 3 109.29 0.33 105.041.04 106.54 0.99 106.29 0.60 105.31 0.85 4 113.13 0.46 109.14 1.15110.09 0.72 110.61 0.41 109.24 0.82 7 124.15 0.70 119.90 1.39 121.291.32 121.59 0.72 117.15 1.36 8 127.82 0.80 123.38 1.52 124.44 1.43124.47 1.24 118.52 1.89 9 132.40 0.80 125.50 1.59 125.91 1.69 125.821.95 118.60 2.62 10 135.91 0.83 123.51 1.77 123.30 2.47 123.87 2.59115.26 3.19 11 140.42 1.13 119.82 1.98 119.55 2.76 121.20 2.99 112.943.48 14 152.59 1.72 111.79 1.40 111.50 1.87 111.80 1.65 108.37 2.75 15151.87 1.87 110.82 1.41 110.63 2.05 110.85 1.44 108.68 2.45 16 156.472.25 111.33 1.74 111.08 2.32 110.98 1.31 109.21 2.16 17 157.65 2.08111.24 1.62 112.06 2.36 111.42 1.66 110.16 2.03 18 161.98 2.71 112.162.21 112.60 2.78 112.54 1.64 111.14 2.11 20 167.36 2.93 113.49 2.37114.17 3.24 114.82 2.12 113.17 2.49 21 168.73 3.07 114.93 2.62 115.253.68 114.76 2.30 113.80 2.68 23 174.51 3.54 116.96 3.02 118.48 3.49116.76 2.51 114.93 2.62 24 176.27 3.50 117.63 3.13 118.58 3.71 116.562.57 114.99 2.51 0.1 mg/kg 0.3 mg/kg 1.0 mg/kg 5.0 mg/kg Day Mean S.E.MMean S.E.M Mean S.E.M Mean S.E.M 0 100.00 0.00 100.00 0.00 100.00 0.00100.00 0.00 1 99.31 0.61 99.26 0.28 98.81 0.27 98.25 0.58 2 102.87 0.73102.98 0.43 103.18 0.50 101.82 0.53 3 106.26 0.82 106.95 0.50 106.520.55 105.47 0.58 4 110.20 0.64 110.50 0.58 110.52 0.67 109.29 0.58 7120.50 1.20 120.03 0.82 121.54 1.15 119.77 1.19 8 123.48 1.58 121.381.31 124.28 1.59 121.96 1.72 9 125.46 2.47 121.57 2.09 125.60 2.23123.04 2.42 10 123.95 3.38 118.27 3.07 124.11 2.97 120.00 2.81 11 121.983.93 116.02 3.32 121.27 3.42 117.97 2.98 14 113.90 2.14 108.43 1.94111.72 2.27 111.58 2.59 15 113.66 1.91 109.59 2.12 112.30 2.23 113.332.37 16 115.06 2.00 111.54 2.02 115.00 2.36 116.06 2.30 17 115.99 2.18113.57 2.04 118.08 2.32 119.14 2.42 18 118.01 2.29 116.13 2.14 121.162.55 122.14 2.61 20 122.17 2.57 120.62 2.20 126.90 2.87 128.60 2.77 21123.49 2.90 122.88 2.49 130.41 2.98 132.82 2.84 23 124.35 3.02 125.362.83 137.81 3.09 140.79 2.83 24 123.77 2.80 125.33 2.75 138.93 2.76141.77 2.61

The effect of treating animals with various doses of anti-NGF antibodyE3 on body weight was statistically analyzed by using two-way ANOVA tocompare the results obtained pairwise between arthritic animals treatedwith vehicle with those treated with a given dose of antibody E3. Usingdata normalized to weight on day 14 (Table 21), doses of 0.03 mg/kg ofE3 resulted in a significant change in body weight (p<0.005). At allhigher dose of E3, the difference between treated and untreatedarthritic animals was significant (p=or <0.0001). Using data normalizedto weight on day 0 (Table 22), dose of 0.03 mg/kg of E3 resulted in asignificant change in body weight (p<0.002). At all higher dose of E3,the difference between treated and untreated arthritic animals wassignificant (p<0.0001).

Again in agreement with earlier studies, rats treated with E3 showedless apparent weight loss than saline treated arthritic rats (Table 22and FIG. 22). In fact, rats treated with high doses of antibody E3 wererecovering the earlier weight loss, and actually gaining weight fasterthan their non-arthritic cohorts (Table 21 and FIG. 21).

Example 9. Analgesic Effects of Anti-NGF Antibody E3 Inpatients withModerate to Severe Pain from Osteoarthritis of the Knee

In this randomized, placebo-controlled, double-blind, dose-escalationstudy, analgesic effects of single intravenous doses (3 μg/kg, 10 μg/kg,30 μg/kg, 100 μg/kg, or 300 μg/kg) of anti-NGF antibody E3 were comparedwith placebo in patients with moderate to severe pain fromosteoarthritis of the knee. Adult males and females (ages between 35 to65) who were experiencing moderate-to-severe pain from osteoarthritis ofthe knee, but were in otherwise overall good health were enrolled in thestudy. During the screening period, patients were required todiscontinue arthritis medications such as NSAIDs, cyclooxygenase type-2(COX-2) inhibitors, and opiates at least 14 days prior to administrationof anti-NGF antibody E3. Patients were allowed to use rescue medicationsof acetaminophen or ibuprofen with some restrictions based on laboratoryfindings or past medical history. Some patients took rescue medicationsduring the study, but rescue medications were not consumed for 2 daysbefore and after administration of anti-NGF antibody E3.

Thirty-four patients were admitted to the study and assessed asinpatients for on Study Days −1, 1 and 2. Anti-NGF antibody E3administration occurred on the morning of Day 1. Electronic diaries wereused to record index knee pain and rescue medication usage in clinic andat home. Ten patients were treated with placebo. Twenty-four patientswere treated with anti-NGF antibody E3: four patients per dose level for3 μg/kg (cohort 1), 10 pg/kg (cohort 2), and 30 μg/kg (cohort 3); andsix patients per dose level for 100 μg/kg (cohort 4) and 300 μg/kg(cohort 5). The placebo used was sterile 0.9% Sodium Chloride Injection,USP (normal saline). Anti-NGF antibody E3 was a frozen liquidformulation consisting of 10 mg/ml antibody in an aqueous solution of 10mM histidine, 275 mM sucrose, 0.01% polysorbate 20, pH 6.0. One hourprior to IV administration, frozen antibody was thawed and diluted intoSodium Chloride Injection, USP. The volume for each patient wascalculated based on the subject's Day −1 weight and assigned dose level,ranging from 3-6 cc for cohort 1, 10-20 cc for cohort 2, 30-60 cc forcohort 3, and 100 cc for cohort 4 and cohort 5. For cohorts 1 and 2,antibody E3 was administered by a slow IV bolus over 3 to 5 minutes,followed with an IV flush of 5 cc Sodium Chloride Injection, USP. Forcohorts 3-5, antibody E3 was administered at 100 cc/hour via infusionpump, followed with an IV flush of 5 cc Sodium Chloride Injection, USPand discontinuation of the IV. Within each cohort, the placebo (SodiumChloride Injection, USP) was administered by slow bolus or continuousinfusion in the same fashion as the antibody E3.

After two in-patient days, patients were discharged home. Depending ofthe dose level cohort, they continued to be assessed for pain levels for28 days after antibody E3 administration (Cohorts 1, 2, and 3) or up to181 days after antibody E3 administration (Cohorts 4 and 5). Periodicsafety assessments was performed for 181 days in all patients.

Analgesic effects were assessed before dosing and at multiple timesafter administration of antibody E3. Validated electronic VAS rangingfrom 0-100 with 101 point resolution was used for current index kneepain. Patients were cued 4 times daily and instructed to indicate thelevel of arthritis pain in the index knee at that moment. Validatedelectronic VAS ranging from 0-100 with 101 point resolution was alsoused for index knee pain during walking. VAS for index knee pain duringwalking was completed daily, concurrent with the fourth current indexknee pain VAS.

Electronic diary for data collection was issued on day −8 and a 7-dayscreening assessment of electronic diary compliance was performed fromday −8 to day −2 to determine success in using the electronic diariesfor a given subject. Subjects were instructed to complete 4 diaries perday. The mean VAS recorded during the screening period is used asbaseline for assessment of efficacy.

WOMAC (Western Ontario and McMaster Universities Arthritis Scale) 3.1Osteoarthritis Index Version VA.1^(©) was also used for assessingarthritis pain. WOMAC 3.1M consists of 24 questions divided into 3domains: pain (5 questions), stiffness (2 questions), and physicalfunction (17 questions). Bellamy et al., J. Rheumatol 15:1833-40 (1988);and Bellamy, Semin. Arthritis Rheum. 18:14-7 (1989). The physicalfunction domain provides information of the ability to performactivities of daily living. Patients performed the test directly on theelectronic diary during designated clinic visits, using validatedelectronic VAS ranging from 0-100 with 101 point resolution. Each domainwas scored by determining the means of the VAS scores of the componentquestions. The total WOMAC 3.1™ was scored by determining the means ofthe scores from each of the 3 domains. Patients were also required toreport rescue medication used via the electronic diary.

Outcome was assessed as change from baseline (pain level duringscreening period), expressed as pain intensity difference (PID) orsummed pain intensity difference (SPID) for different dose levels.Changes from baseline in the activity measurements were analyzed usingan analysis of covariance (ANCOVA) model with treatment as the mainfactor and baseline pain as a covariate. For means and mean changes,comparisons of antibody E3 with placebo was made using Dunnett's test.Tukey-Ciminera-Heyse trend test was used to assess the dose-responserelationship.

As shown in FIG. 24, decrease of mean daily pain intensity after thesingle administration of anti-NGF antibody E3 was observed in every dosetested. Generally, the effect in reducing pain intensity lasted longerin the higher dose (30 μg/kg, 100 μg/kg and 300 μg/kg) treated groupthan the lower dose (3 μg/kg and 10 μg/kg) treated group. As shown inFIG. 25, the pain intensity reductions lasted for at least 80 days afteradministration of 100 μg/kg of E3.

Table 23 below shows the summed pain intensity differences (SPID) foreach dosing of E3 for days 2-8, days 2-14, days 2-21, and days 2-28 ascompared to baseline levels. ANCOVA was used for statistically analysis.Table 23 indicates that pain reduction was statistically significant(p<0.05) after single administration of anti-NGF antibody E3.

TABLE 23 Summed pain intensity differences for varying time intervals ascompared to baseline. Days 2-8 Days 2-14 Days 2-21 Days 2-28 Placebo−269  −486  −758 −1055  3 μg/kg −572  −956 −1048 −1067 (0.11) (0.18)(0.63) (0.99) (0.43) (0.62) (0.99) (1.00)  10 μg/kg −671 −1174 −1623−2020 (0.04) (0.05) (0.15) (0.29) (0.17) (0.24) (0.55) (0.81)  30 μg/kg−733 −1371 −2042 −2566 (0.02) (0.02) (0.04) (0.10) (0.09) (0.08) (0.17)(0.40) 100 μg/kg −766 −1403 −1996 −2726 (<0.01) (<0.01) (0.02) (0.04)(0.02) (0.02) (0.10) (0.17) 300 μg/kg −769 −1340 −1869 −2521 (<0.01)(<0.01) (0.04) (0.07) (0.02) (0.04) (0.17) (0.29) All −713 −1269 −1752−2254 (<0.01) (<0.01) (0.01) (0.04) (<0.01) (<0.01) (0.06) (0.18)Statistically analysis was performed with no adjustment and withadjustment for multiple comparisons. The first row of the p values inparenthesis for each time interval was without adjustment; the secondrow of p values in parenthesis was with Dunnett's adjustment.

As shown in FIG. 26, percent of maximal reduction in SPID by the singleadministration of anti-NGF antibody E3 reached about 45% to about 65%from day 2 to day 14 for every E3 dose tested, and reached about 45% toabout 60% from day 2 to day 28 for 10 μg/kg, 30 μg/kg, 100 μg/kg, and300 μg/kg E3 dose.

As shown in FIG. 27, administration of anti-NGF antibody E3 also reducedWOMAC score from day 1 to day 28. Pain, physical function and stiffnessscore were reduced significantly at 100 μg/kg dose of E3. These dataindicate that single administration of anti-NGF antibody not onlyreduces pain, but also improves physical function and stiffness inpatients having osteoarthritis.

Deposit of Biological Material

The following materials have been deposited with the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va., USA(ATCC):

ATCC Accession Date of Material No. Deposit Eb.911.3E E3 light chainPTA-4893 Jan. 8, 2003 Eb.pur.911.3E E3 light chain PTA-4894 Jan. 8, 2003Db.911.3E E3 heavy chain PTA-4895 Jan. 8, 2003

Vector Eb.911.3E is a polynucleotide encoding the E3 light chainvariable region; vector Eb.pur.911.3E is a polynucleotide encoding E3light chain variable region, and vector db.911.3E is a polynucleotideencoding the E3 heavy chain variable region.

This deposit was made under the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Rinat Neuroscience Corp. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC Section 122 and the Commissioner's rulespursuant thereto (including 37 CFR Section 1.14 with particularreference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Antibody sequences Heavy chain variable region (Kabat CDRs areunderlined; Chothia CDRs are 

 

) (SEQ ID NO: 1) QVQLQESGPGLVKPSETLSLTCTVSGFSLI

WIRQPPGKGLEWIG

RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR

WGQGTLVTVS Light chain variable region (Kabat CDRs areunderlined; Chothia CDRs are 

 

) (SEQ ID NO: 2) DIQMTQSPSSLSASVGDRVTITC

WYQQKPGKAPKLLIY

GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC

F GQGTKLEIKRT E3 heavy chain extended CDRs CDRH1: (SEQ ID NO: 3)GFSLIGYDLN CDRH2: (SEQ ID NO: 4) IIWGDGTTDYNSAVKS CDRH3: (SEQ ID NO: 5)GGYWYATSYYFDY E3 light chain extended CDRs CDRL1: (SEQ ID NO: 6)RASQSISNNLN CDRL2: (SEQ ID NO: 7) YTSRFHS CDRL3: (SEQ ID NO: 8)QQEHTLPYT Mouse monoclonal antibody 911 extended CDRs911 heavy chain extended CDRs CDRH1: (SEQ ID NO: 9) GFSLIGYDIN CDRH2:(SEQ ID NO: 10) MIWGDGTTDYNSALKS CDRH3: (SEQ ID NO: 11) GGYYYGTSYYFDY911 light chain extended CDRs CDRL1: (SEQ ID NO: 12) RASQDISNHLN CDRL2:(SEQ ID NO: 13) YISRFHS CDRL3: (SEQ ID NO: 14) QQSKTLPYTE3 heavy chain amino acid sequence (full) (SEQ ID NO: 16)QVQLQESGPGLVKPSETLSLTCTVSGFSLIGYDLNWIRQPPGKGLEWIGIIWGDGTTDYNSAVKSRVTISKDTSKNQFSLKLSSVTAADTAVYYCARGGYWYATSYYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK3E light chain amino acid sequence (full antibody) (SEQ ID NO: 17)DIQMTQSPSSLSASVGDRVTITCRASQSISNNLNWYQQKPGKAPKLLIYYTSRFHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQEHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHXVYACEV THQGLSSPVTKSFNRGEC3E heavy chain nucleotide sequence (full antibody) (SEQ ID NO: 65)CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCACCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTMTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGT CCCTGTCTCCAGGAAAGTAA3E heavy chain variable domain nucleotide sequence (SEQ ID NO: 66)CAGGTGCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCCGAGACCCTGTCCCTCACCTGCACTGTCTCTGGGTTCTCACTTATCGGCTATGATCTTAACTGGATCCGACAGCCTCCAGGGAAGGGACTGGAGTGGATTGGGATTATCTGGGGTGATGGAACCACAGACTATAATTCAGCTGTCAAATCCCGCGTCACCATCTCAAAAGACACCTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGGAGGTTATTGGTACGCCACTAGCTACTACTTTGACTACTGGGGCCAGGGCA CCCTGGTCACCGTCTCCTCA3E light chain nucleotide sequence (full antibody) (SEQ ID NO: 67)GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGCACTGTGGCTGCACCATCTGTGTTCATCTTTCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCACGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACMAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTG AGTGCTAA3E light chain variable domain nucleotide sequence (SEQ ID NO: 68)GATATCCAGATGACACAGTCCCCATCCTCCCTGTCTGCCTCTGTGGGTGACCGCGTCACCATCACCTGCCGCGCATCTCAGTCCATTAGCAATAATCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCCCCAAAACTCCTGATCTACTACACCTCACGCTTCCACTCAGGTGTCCCATCACGCTTCAGTGGCAGTGGCTCTGGTACAGATTTCACCTTCACCATTAGCAGCCTGCAACCAGAAGATATTGCCACTTATTACTGCCAACAGGAGCATACCCTTCCATATACCTTCGGTCAAGGCACCAAGCTGGAGATCAAACGC

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

1-85. (canceled)
 86. A method for treating pain, improving physicalfunction and improving stiffness in an individual having osteoarthritiscomprising administering an effective amount of an NGF antagonist to theindividual.
 87. The method of claim 86, wherein the individual is ahuman.
 88. The method of claim 86, wherein the NGF antagonist is ananti-NGF antagonist antibody.
 89. The method of claim 86, wherein theanti-NGF antagonist antibody is administered at a dosing frequency in arange from once every week to once every twelve weeks.
 90. The method ofclaim 86, wherein the anti-NGF antagonist antibody is administered onceevery month, once every two months, once every three months, once everyfour months, once every five months, or once every six months.
 91. Themethod of claim 86, wherein the anti-NGF antagonist antibody isadministered once every three months.
 92. The method of claim 86,wherein the anti-NGF antagonist antibody is administered at a dose in arange from about 3 μg/kg to about 1 mg/kg.
 93. The method of claim 92,wherein the anti-NGF antagonist antibody is administered at a dose ofabout 100 μg/kg.
 94. The method of claim 92, wherein the anti-NGFantagonist antibody is administered at a dose of about 300 μg/kg. 95.The method of claim 86, wherein the anti-NGF antagonist antibody isadministered intravenously.
 96. The method of claim 86, wherein theanti-NGF antagonist antibody is administered subcutaneously.
 97. Themethod of claim 86, wherein the anti-NGF antagonist antibody is amonoclonal antibody.
 98. The method of claim 86, wherein the anti-NGFantagonist antibody is a humanized antibody.
 99. The method of claim 86,wherein the anti-NGF antagonist antibody binds human NGF.
 100. Themethod of claim 99, wherein the anti-NGF antagonist antibody furtherbinds rodent NGF.
 101. The method of claim 88, wherein the anti-NGFantagonist antibody: (a) binds NGF with a K_(D) of less than about 2 nM;(b) inhibits human NGF-dependent survival of mouse E13.5 trigeminalneurons with an IC50 of about 100 pM or less, wherein the IC50 ismeasured in the presence of about 15 pM human NGF; and (c) inhibitshuman NGF-dependent survival of mouse E13.5 trigeminal neurons with anIC50 of about 10 pM or less, wherein the IC50 is measured in thepresence of about 1.5 pM of NGF.
 102. The method of claim 88, whereinthe anti-NGF antagonist antibody comprises a heavy chain variable regioncomprising: (a) a CDR1 region shown in SEQ ID NO: 3; (b) a CDR2 regionshown in SEQ ID NO:4; and (c) a CDR3 region shown in SEQ ID NO:
 5. 103.The method of claim 88, wherein the anti-NGF antagonist antibodycomprises a light chain variable region comprising: (a) a CDR1 regionshown in SEQ ID NO:6; (b) a CDR2 region shown in SEQ ID NO:7; and (c) aCDR3 region shown in SEQ ID NO:
 8. 104. The method of claim 103, whereinthe anti-NGF antagonist antibody further comprises a heavy chainvariable region comprising: (a) a CDR1 region shown in SEQ ID NO: 3; (b)a CDR2 region shown in SEQ ID NO:4; and (c) a CDR3 region shown in SEQID NO:
 5. 105. The method of claim 86, wherein the anti-NGF antagonistantibody is an antibody comprising the amino acid sequences shown in SEQID NOS: 1 and
 2. 106. The method of claim 105, wherein the anti-NGFantagonist antibody is an antibody comprising the amino acid sequencesshown in SEQ ID NOS: 16 and 17.